Antibiotic sensitivity-restoring and photosensitive agents

ABSTRACT

The present disclosure describes a method to treat conditions, including bacterial infections and cancer, using a photosensitive compound that, upon exposure to white light, can be activated. The photosensitive compound can also interact synergistically with antibiotics used concomitantly to kill drug-resistant bacteria. The photosensitive compounds can also be used to inhibit the proliferation of cancer cells.

CROSS REFERENCE

This application is a continuation of U.S. application Ser. No.15/798,545 filed Oct. 31, 2017 which is a continuation of U.S.application Ser. No. 15/133,430, filed Apr. 20, 2016, which issued asU.S. Pat. No. 9,834,514 on Dec. 5, 2017, which claims the benefit ofU.S. Provisional Application No. 62/149,738, filed Apr. 20, 2015, andU.S. Provisional Application No. 62/306,165, filed Mar. 10, 2016, eachof which is incorporated herein by reference in its entirety.

GOVERNMENT RIGHTS

The invention was made with government support under RR016480-12 by theNational Center for Research Resources and under GM103451-12 by theNational Institutes of Health. The government has certain rights in theinvention.

BACKGROUND

Antibiotics have become a mainstay of anti-microbial therapy, especiallyin treatment of bacterial infections. However, overuse of antibioticshas led to the emergence of drug-resistant bacteria due to antibioticeffectiveness and ease of access. The pathogenic bacteria that wereinitially sensitive to specific antibiotics are rapidly evolving toevade targeting by antibiotics. The development of a therapy that cancombat the emergence of drug-resistant bacteria can assist incontrolling the widespread evolution of pathogenic microbes.

INCORPORATION BY REFERENCE

Each patent, publication, and non-patent literature cited in theapplication is hereby incorporated by reference in its entirety as ifeach was incorporated by reference individually.

SUMMARY OF THE INVENTION

In some embodiments, the invention provides a method of treating acondition, the method comprising administering to a subject in needthereof a therapeutically-effective amount of a compound that binds abiological structure, thereby decreasing drug resistance in a cell, anda therapeutically-effective amount of a second agent.

In some embodiments, the invention provides a compound of the formula:

wherein:

-   -   R¹ is hydrogen or an ester group;    -   R² is hydrogen, halogen, or L¹-Ar¹;    -   R³ is hydrogen, halogen, or L²-Ar²;    -   or R² and R³ together with the atoms to which R² and R³ are        bound form a substituted or unsubstituted ring;    -   each L¹ and L² is independently a linking group or a bond;    -   each Ar¹ is a substituted or unsubstituted aryl group;    -   each Ar² is a substituted or unsubstituted aryl group wherein        Ar² is not substituted with an amide, amine, nitro, imine, or        ester group;    -   each A¹, A², A³, and A⁴ is independently C(R^(1a)),        C(R^(1a))(R^(1b)), N, or N(R^(1a));    -   each R^(1a) and R^(1b) is independently hydrogen, halogen,        hydroxyl, sulfhydryl, nitro, nitroso, cyano, azido, a sulfoxide        group, a sulfone group, a sulfonamide group, a sulfonic acid        group, an imine group, an acyl group, an acyloxy group, alkyl,        alkenyl, alkynyl, an alkoxy group, an ether group, a carboxylic        acid group, a carboxaldehyde group, an ester group, an amine        group, an amide group, a carbonate group, a carbamate group, a        thioether group, a thioester group, a thioacid group, aryl,        aryloxy, arylalkyl, arylalkoxy, heterocyclyl, heterocyclylalkyl,        heteroaryl, or heteroarylalkyl, any of which is substituted or        unsubstituted; and    -   each        is independently a single or double bond,        or a pharmaceutically-acceptable salt thereof, wherein the        compound is not:

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts a fluorescent binding assay for a compound of theinvention.

FIG. 2 shows a competition assay between compound 1 and INF-55.

FIG. 3 shows a competition assay between compound 1 and reserpine.

FIG. 4 shows an image of a culture dish (left) and a scanning electronmicroscope image (right) of MRSA upon receiving photodynamic therapyusing a hand-held UV flashlight.

FIG. 5 depicts a chessboard bacterial patterning system forvisualization of bacterial growth.

FIG. 6 depicts light-activated killing of hospital-acquired MRSA withcompound 1.

FIG. 7 depicts light-activated killing of hospital-acquired MRSA withcompound 1.

FIG. 8 depicts a UV/Vis spectrum of compound 1.

FIG. 9 depicts light-activated killing of community-acquired MRSA withcompound 1.

FIG. 10 depicts light-activated killing of S. aureus with compound 1.

FIG. 11 depicts light-activated killing of VRE with compound 1.

FIG. 12 depicts light-activated killing of S. pyogenes with compound 1.

FIG. 13 depicts light-activated killing of S. mutans with compound 1.

FIG. 14 represents an isobologram for synergy between compound 1 andvarious antibiotics.

FIG. 15 represents a zoomed-in portion of the isobologram from FIG. 14.

FIG. 16 illustrates the effect of treatment of A. baumannii withcompound 1 and polymyxin B.

FIG. 17 illustrates the effect of treatment of E. coli with compound 1and polymyxin B.

FIG. 18 illustrates the effect of treatment of MRSA with compound 1 andtetracycline.

FIG. 19 illustrates the effect of treatment of MRSA with compound 1 anddoxycycline.

FIG. 20 illustrates the effect of treatment of MRSA with compound 1 andnorfloxacin.

FIG. 21 illustrates the effect of treatment of MRSA with compound 1 anddicloxicillin.

FIG. 22 illustrates the effect of treatment of MRSA with compound 1 andoxacillin.

FIG. 23 illustrates the effect of treatment of MRSA with compound 1 andpenicillin G.

FIG. 24 illustrates the effect of treatment of MRSA with compound 1 andtobramycin.

FIG. 25 represents the MIC of vancomycin in presence or absence ofcompound 1 against MRSA.

FIG. 26 illustrates the effect of treating P. aeruginosa with compound 1and polymyxin E.

FIG. 27 illustrates the effect of treating P. aeruginosa with compound 7and polymyxin E.

FIG. 28 illustrates the effect of treating P. aeruginosa with compound 8and polymyxin E.

FIG. 29 illustrates the effect of treating P. aeruginosa with compound 9and polymyxin E.

FIG. 30 illustrates the effect of treating P. aeruginosa with compound10 and polymyxin E.

FIG. 31 illustrates the effect of treating P. aeruginosa with compound11 and polymyxin E.

FIG. 32 depicts in image of a turbidity comparison of MRSA isolatestreated with compound 7 and/or oxacillin.

FIG. 33 depicts the quantification of the MRSA isolates shown in FIG. 32upon being treated with compound 7 and/or oxacillin.

FIG. 34 illustrates the effect of treating MRSA with compound 1 andnorfloxacin or oxacillin.

FIG. 35 depicts the synergistic effect of treating MRSA with compound 7and oxacillin or norfloxacin.

FIG. 36 depicts the synergistic effect of treating MRSA with compound 8and oxacillin or norfloxacin.

FIG. 37 depicts the synergistic effect of treating MRSA with compound 9and oxacillin or norfloxacin.

FIG. 38 depicts the synergistic effect of treating MRSA with compound 10and oxacillin or norfloxacin.

FIG. 39 depicts the synergistic effects of treating MRSA with compound10 and norfloxacin or oxacillin.

FIG. 40 depicts the synergistic effect of treating MRSA with compound 11and oxacillin or norfloxacin.

FIG. 41 depicts the synergistic effect of treating MRSA with compound 11and norfloxacin or oxacillin.

FIG. 42 depicts the synergistic effect of treating VRE with compound 7with vancomycin, tetracycline or norfloxacin.

FIG. 43 depicts the synergistic effect of treating VRE with compound 8with vancomycin, tetracycline or norfloxacin.

FIG. 44 depicts the synergistic effect of treating VRE with compound 9with tetracycline or norfloxacin.

FIG. 45 depicts the synergistic effect of treating VRE with compound 10with vancomycin, tetracycline, dicloxicillin or norfloxacin.

FIG. 46 depicts the synergistic effect of treating VRE with compound 11with vancomycin, tetracycline or norfloxacin.

FIG. 47 depicts the synergistic effect of treating CRE with compounds7-11 with Polymyxin B.

FIG. 48 illustrates the time-dependent effects of treating CRE withcompounds 8 or 9 with and without PME.

FIG. 49 illustrates the time-dependent effects of treating CRE withcompounds 10 or 11 with and without PME.

FIG. 50 illustrates changes in MICs of compounds 7 and 9 over timeagainst A. baumannii in the presence and absence of PME.

FIG. 51 illustrates changes in MIC of compound 8 over time against A.baumannii in the presence and absence of PME and PMB.

FIG. 52 illustrates changes in MICs of compounds 10 and 11 over timeagainst A. baumannii in the presence and absence of PME.

FIG. 53 illustrates the effect of compound 1 and light versus polymyxinB in treatment of A. baumannii.

FIG. 54 illustrates the effect of compound 1 and light versus polymyxinB in treatment of E. coli.

FIG. 55 illustrates the effect of treating P. aeruginosa withcombinations of compound 1, PME, and light.

FIG. 56 illustrates production of singlet oxygen using compound 1.

FIG. 57 displays an image of minimum inhibitory concentrationmeasurements of compounds 7-10 against S. aureus.

FIG. 58 displays an image of final minimum inhibitory concentrationmeasurements of compounds 7-10 when used to treat S. aureus uponreceiving 30 exposures.

DETAILED DESCRIPTION Antibiotics.

Antibiotics are used globally as therapy in the treatment of, forexample, bacterial infections. Antibiotics can also be effective againstsome fungi and protozoa. Antibiotics can be classified asbacteriostatic, wherein the antibiotic inhibits reproduction of thebacteria, and bactericidal, wherein the antibiotic kills the bacteria.Antibiotics can be further classified by mechanism of action, which caninclude, for example, inhibition of bacterial cell wall synthesis,inhibition of bacterial cell membrane synthesis, inhibition of essentialbacterial enzymes, inhibition of cell division, inhibition ofpeptidoglycan synthesis, inhibition of protein synthesis via binding toa 30S or 50S subunit of bacterial ribosome, inhibition of isoprenylpyrophosphate, inhibition of folate synthesis, and production of toxicfree radicals.

Antibiotics can be used to treat bacterial infections. Antibiotics canalso be used prophylactically for a subject, for example, having a woundthat is likely to become infected, a subject about to undergo surgery, asubject about to receive dental treatment, or a subject who suffers fromrecurring infections including, for example, cellulitis, urinary tractinfections, and rheumatic fever.

Overuse of antibiotics in the healthcare and agricultural industries,and misuse of antibiotics, including use of antibiotics in the treatmentof viral infections, cessation of antibiotic therapy prior to end ofprescribed period, and prophylactic use of antibiotics by travelers, hasled to the emergence of drug-resistant bacteria. Mutations that can helpbacteria survive treatment with an antibiotic can quickly becomeprevalent throughout a bacterial population, and genetic elementsencoding resistance mechanisms can be transferred between bacterialspecies.

Mechanisms of Bacterial Resistance to Antibiotics.

Bacteria can use various mechanisms to avoid killing by an antibiotic.Bacteria can, for example, modify the protein targeted by theantibiotic, enzymatically inactivate the antibiotic, decrease theability of the antibiotic to enter the cell, transfer resistance genesbetween organisms via conjugation, transduction, or transformation, orincrease the exit of the antibiotic from the cell using efflux pumps.

Efflux pumps are transport proteins found in both Gram-positive andGram-negative bacteria. Five major classes of efflux pumps can exist inprokaryotes including, for example, major facilitator (MF), multidrugand toxic efflux (MATE), resistance-nodulation-division (RND), smallmultidrug resistance (SMR), and ATP binding cassette (ABC). Efflux pumpscan be specific for a single substrate or transport a range ofstructurally-similar or -dissimilar compounds, including antibiotics.Increased expression of efflux pumps can be correlated with resistanceto associated substrates. Efflux pumps can also be used to transport,for example, toxins, metabolites, drugs, lipophilic cationic drugs, bileacids, fatty acids, and lipids.

One mechanism that can be employed by drug-resistant bacteria ismulti-drug efflux via membrane transporter proteins known as multidrugefflux systems (MES), which can recognize more than one substrate. TheMES can be classified as, for example, ABC, MATE, RND, SMR, and themultiantimicrobial extrusion protein family. In Gram-positive bacteria,the major efflux systems are the chromosomally encoded Major FacilitatorSuperfamily (MFS), Nor-family (NorA, NorB, NorC), and the MdeA the MATEmepRAB (multidrug export protein and the SMR SepA) family. TheGram-positive efflux systems can have overlapping specificities and canaccept a large variety of structurally unrelated antibiotics including,for example, quinolones, tetracyclines, and monovalent and divalentantimicrobial cations, which can include intercalating dyes, quaternaryammonium compounds, diamidines, biguanidines, and plant secondarymetabolites.

Non-limiting examples of efflux systems in Gram-negative bacteriainclude CraA and AmvA, which mediate antimicrobial and disinfectantresistance in A. baumannii, and MdfA of E. coli. The MFS and ABC effluxsystem super families can be commonly found among resistant strains ofmycobacteria, including Mycobacterium tuberculosis.

The development of new strategies to target drug-resistant bacteria canbe applied to especially-virulent strains of bacteria, which can causesevere and widespread infection in nursing homes and hospitals, and arenot susceptible to standard antibiotic treatment. For example,Staphylococcus aureus can be a dangerous and versatile opportunisticpathogen. S. aureus can cause, for example, superficial skin infectionsresulting from cuts, abrasions, turf burns, and severe invasivediseases. Originally responsive to penicillin, a number of S. aureusstrains are now resistant to various classes of antibiotics including,for example, β-lactams, macrolides, and vancomycin.

Methicillin-resistant (MRSA) and vancomycin-resistant S. aureus (VRSA)are a significant health threat and constitute a major cause ofmortality from bacterial infections. Several chromosomally-encodedefflux systems can be present in MRSA including, for example, NorA,NorB, NorC, MepA, MdeA, SepA, SdrM, and LmrS. LmrS can expel, forexample, ampicillin. Some of the plasmid-mediated efflux systems foundin MRSA can include, for example, QacA, QacB, Smr, QacG, QacH, and QacJ.Some strains of MRSA can carry more than one efflux system.

Compounds of the Invention.

The present compounds can potentiate the effects of many functionally-and mechanistically-diverse antibiotics against, for example, MRSA,which displays efflux-mediated resistance to antibiotics. The presentcompounds can display synergy with antibiotics including, for example,polymyxin B (PMB) against Gram-negative bacterial strains.

Compounds of the invention can increase the potency of antibiotics. Forexample, the mean inhibitory concentrations (MIC) for a compound of theinvention and ampicillin were determined to be about 200 μM and about4579 μM, respectively. When the compound was used in combination withampicillin, the concentration of the compound needed for full killingwas 3 μM, a 127-fold decrease in concentration, and the concentration ofampicillin needed was reduced to about 572 μM, an 8-fold decrease inconcentration. The ability of compounds of the invention to synergizewith structurally and mechanistically unrelated antibiotics can be aresult of the inhibition of bacterial efflux. Thus, the discoveredpotentiation of antibiotic activity with2,3-di-((E)-2-arylethenyl)indoles can restore the activity ofantibiotics rendered inactive against MRSA, or other bacterial strains,due to the increased presence of bacterial efflux pumps.

Non-limiting examples of compounds of the invention include compounds ofany of the following formulae:

or a pharmaceutically-acceptable salt thereof, wherein: RING is a ringsystem; each of Cy¹, Cy², Cy³, and Cy⁴ is independently a cyclic group;and each of L¹, L², L³, and L⁴ is independently a linking group.

Non-limiting examples of cyclic groups or of a ring system includearomatic, non-aromatic, heterocyclic, carbocyclic, monocyclic, andpolycyclic groups. A polycyclic group can be, for example, bicyclic,tricyclic, or tetracyclic. A polycyclic group can be, for example,fused, bridged, or spiro, or any combination thereof. Non-limitingexamples of aromatic groups include heterocyclic, carbocyclic,monocyclic, and polycyclic rings. Any such group can be substituted orunsubstituted at any position, with any number of substituents.Non-limiting examples of substituents include: halogens, hydroxylgroups, sulfhydryl groups, amino groups, nitro groups, nitroso groups,cyano groups, azido groups, sulfoxide groups, sulfone groups,sulfonamide groups, carboxyl groups, carboxaldehyde groups, iminegroups, alkyl groups, halo-alkyl groups, alkenyl groups, halo-alkenylgroups, alkynyl groups, halo-alkynyl groups, alkoxy groups, aryl groups,aryloxy groups, aralkyl groups, arylalkoxy groups, heterocyclyl groups,acyl groups, acyloxy groups, carbamate groups, amide groups, epoxides,ester groups, and any other substituent described herein.

A linking group can be any chemical group that attaches groups of thestructure together. A linking group can comprise, for example, analkylene group, an alkenylene group, an alkynylene group, a polyether,such as polyethylene glycol (PEG), a polyester, a polyamide, or apolyamine, any of which being unsubstituted or substituted with anynumber of substituents, such as halogens, hydroxyl groups, sulfhydrylgroups, amino groups, nitro groups, nitroso groups, cyano groups, azidogroups, sulfoxide groups, sulfone groups, sulfonamide groups, carboxylgroups, carboxaldehyde groups, imine groups, alkyl groups, halo-alkylgroups, alkenyl groups, halo-alkenyl groups, alkynyl groups,halo-alkynyl groups, alkoxy groups, aryl groups, aryloxy groups, aralkylgroups, arylalkoxy groups, heterocyclyl groups, acyl groups, acyloxygroups, carbamate groups, amide groups, epoxides, ester groups, and anyother substituent described herein.

Non-limiting examples of compounds of the invention include compounds ofany of the following formulae:

or a pharmaceutically-acceptable salt thereof, wherein: X is N, NH,NR^(N), S, or O; each

is independently a single bond or a double bond; R^(N) is hydroxyl,sulfhydryl, nitro, nitroso, cyano, azido, a sulfoxide group, a sulfonegroup, a sulfonamide group, a sulfonic acid group, an imine group, anacyl group, an acyloxy group, alkyl, alkenyl, alkynyl, an alkoxy group,an ether group, a carboxylic acid group, a carboxaldehyde group, anester group, an amine group, an amide group, a carbonate group, acarbamate group, a thioether group, a thioester group, a thioacid group,aryl, aryloxy, arylalkyl, arylalkoxy, heterocyclyl, heterocyclylalkyl,heteroaryl, or heteroarylalkyl, any of which is substituted orunsubstituted, or H; R¹ is H or -L¹-Cy¹; R² is H or -L²-Cy²; R³ is H or-L³-Cy³; R⁴ is H or -L⁴-Cy⁴; or R¹ and R² together with the atoms towhich R¹ and R² are bound form a ring; R² and R³ together with the atomsto which R² and R³ are bound form a ring; or R³ and R⁴ together with theatoms to which R³ and R⁴ are bound form a ring; or R¹ and R² togetherwith the atoms to which R¹ and R² are bound form a first ring and R³ andR⁴ together with the atoms to which R³ and R⁴ are bound form a secondring; each of L¹, L², L³, and L⁴ is independently a linking group; andeach of Cy¹, Cy², Cy³, and Cy⁴ is independently a cyclic group.

Non-limiting examples of compounds of the invention include compounds ofany of the following formulae:

or a pharmaceutically-acceptable salt thereof, wherein: X is N, NH,NR^(N), S, or O; each of Q¹, Q², Q³, and Q⁴ is independently a ringsystem; R¹ is H or -L¹-Cy¹; R² is H or -L²-Cy²; and R⁴ is H or -L⁴-Cy⁴.

Non-limiting examples of compounds of the invention include compounds ofany of the following formulae:

or a pharmaceutically-acceptable salt thereof, wherein: X is N, NH,HR^(N), S, or O; each

is independently a single bond or a double bond; R^(N) is hydroxyl,sulfhydryl, nitro, nitroso, cyano, azido, a sulfoxide group, a sulfonegroup, a sulfonamide group, a sulfonic acid group, an imine group, anacyl group, an acyloxy group, alkyl, alkenyl, alkynyl, an alkoxy group,an ether group, a carboxylic acid group, a carboxaldehyde group, anester group, an amine group, an amide group, a carbonate group, acarbamate group, a thioether group, a thioester group, a thioacid group,aryl, aryloxy, arylalkyl, arylalkoxy, heterocyclyl, heterocyclylalkyl,heteroaryl, or heteroarylalkyl, any of which is substituted orunsubstituted, or H; R¹ is H or -L¹-Cy¹; R² is H or -L²-Cy²; A¹ isC(R^(1a)), C(R^(1a))(R^(1b)), N, or N(R^(1a)); A² is C(R^(2a)),C(R^(2a))(R^(2b)), N, or N(R^(2a)); A³ is C(R^(3a)), C(R^(3a))(R^(3b)),N, or N(R^(3a)); A⁴ is C(R^(4a)), C(R^(4a))(R^(4b)), N, or N(R^(4a));each R^(1a), R^(1b), R^(2a), R^(2b), R^(3a), R^(3b), R^(4a), and R^(4b)is independently: halogen, hydroxyl, sulfhydryl, nitro, nitroso, cyano,azido, a sulfoxide group, a sulfone group, a sulfonamide group, asulfonic acid group, an imine group, an acyl group, an acyloxy group,alkyl, alkenyl, alkynyl, an alkoxy group, an ether group, a carboxylicacid group, a carboxaldehyde group, an ester group, an amine group, anamide group, a carbonate group, a carbamate group, a thioether group, athioester group, a thioacid group, aryl, aryloxy, arylalkyl, arylalkoxy,heterocyclyl, heterocyclylalkyl, heteroaryl, or heteroarylalkyl, any ofwhich is substituted or unsubstituted, or H, or absent, or R^(1a) andR^(1b) together form a carbonyl, a thiocarbonyl, an imine, or an olefin,or R^(2a) and R^(2b) together form a carbonyl, a thiocarbonyl, an imine,or an olefin, or R^(3a) and R^(3b) together form a carbonyl, athiocarbonyl, an imine, or an olefin, or R^(4a) and R^(4b) together forma carbonyl, a thiocarbonyl, an imine, or an olefin; each of L¹ and L² isindependently a linking group; and each of Cy¹ and Cy² is independentlya cyclic group.

In some embodiments, a bond of the compound, such as the bond connectingA¹ and A², the bond connecting A² and A³, or the bond connecting A³ andA⁴, is fused to an additional ring system.

Non-limiting examples of compounds of the invention include compounds ofany of the following formulae:

or a pharmaceutically-acceptable salt thereof, wherein: each Y¹, Y², Z¹,and Z² is independently: a bond, an alkylene group, an alkenylene group,an alkynylene group, an amino linkage, and ether linkage, a thioetherlinkage, an ester linkage, a thioester linkage, an amide linkage, acarbamate linkage, a carbonate linkage, a ureido linkage, a sulfoxidelinkage, a sulfone linkage, a

sulfonamide linkage, or an imine linkage; each

is independently a single, double, or triple bond; and all othervariables are as described previously.

Non-limiting examples of a cyclic group, such as Cy¹ or Cy², includegroups of any of the following formulae:

wherein: each

is independently a single bond or a double bond; each D¹ isindependently

C(R^(D1a)), C(R^(D1a))(R^(D1b)), N, or N(R^(D1a)); each D² isindependently C(R^(D2a)), C(R^(D2a))(R^(D2b)), N, or N(R^(D2a)); each D³is independently C(R^(D3a)), C(R^(D3a))(R^(D3b)), N, or N(R^(D3a)); eachD⁴ is independently C(R^(D4a)), C(R^(D4a))(R^(D4b)), N, or N(R^(D4a));each D⁵ is independently C(R^(D5a)), C(R^(D5a))(R^(D5b)), N, orN(R^(D5a)); each D⁶ is independently C(R^(D6a)), C, or N; each R^(D1a),R^(D1b), R^(D2a), R^(D2b), R^(D3a), R^(D3b), R^(D4a), R^(D4b), R^(D5a),R^(D5b), and R^(D6a) is independently: halogen, hydroxyl, sulfhydryl,nitro, nitroso, cyano, azido, a sulfoxide group, a sulfone group, asulfonamide group, a sulfonic acid group, an imine group, an acyl group,an acyloxy group, alkyl, alkenyl, alkynyl, an alkoxy group, an ethergroup, a carboxylic acid group, a carboxaldehyde group, an ester group,an amine group, an amide group, a carbonate group, a carbamate group, athioether group, a thioester group, a thioacid group, aryl, aryloxy,arylalkyl, arylalkoxy, heterocyclyl, heterocyclylalkyl, heteroaryl, orheteroarylalkyl, any of which is substituted or unsubstituted, or H, orabsent, or R^(D1a) and R^(D1b) together form a carbonyl, a thiocarbonyl,an imine, or an olefin, or R^(D2a) and R^(D2b) together form a carbonyl,a thiocarbonyl, an imine, or an olefin, or R^(D3a) and R^(D3b) togetherform a carbonyl, a thiocarbonyl, an imine, or an olefin, or R^(D4a) andR^(D4b) together form a carbonyl, a thiocarbonyl, an imine, or anolefin, or R^(D5a) and R^(D5b) together form a carbonyl, a thiocarbonyl,an imine, or an olefin.

In some embodiments, a bond of the compound, such as the bond connectingD¹ and D², the bond connecting D² and D³, the bond connecting D³ and D⁴,or the bond connecting D⁴ and D⁵, is fused to an additional ring system.

Non-limiting examples of a cyclic group, such as Cy¹ or Cy², includegroups of any of the following formulae:

wherein: each

is independently a single bond or a double bond; each E¹ isindependently C(R^(E1a)), C(R^(E1a))(R^(E1b)), N, N(R^(E1a)), S, or O;each E² is independently C(R^(E2a)), C(R^(E2a))(R^(E2b)), N, N(R^(E2a)),S, or O; each E³ is independently C(R^(E3a)), C(R^(E3a))(R^(E3b)), N,N(R^(E3a)), S, or O; each E⁴ is independently C(R^(E4a)),C(R^(E4a))(R^(E4b)), N, N(R^(E4a)), S, or O; each E⁵ is independentlyC(R^(E5a)), C, or N; each R^(E1a), R^(E1b), R^(E2a), R^(E2b), R^(E3a),R^(E3b), R^(E4a), R^(E4b), and R^(E5a) is independently: halogen,hydroxyl, sulfhydryl, nitro, nitroso, cyano, azido, a sulfoxide group, asulfone group, a sulfonamide group, a sulfonic acid group, an iminegroup, an acyl group, an acyloxy group, alkyl, alkenyl, alkynyl, analkoxy group, an ether group, a carboxylic acid group, a carboxaldehydegroup, an ester group, an amine group, an amide group, a carbonategroup, a carbamate group, a thioether group, a thioester group, athioacid group, aryl, aryloxy, arylalkyl, arylalkoxy, heterocyclyl,heterocyclylalkyl, heteroaryl, or heteroarylalkyl, any of which issubstituted or unsubstituted, or H, or R^(E1a) and R^(E1b) together forma carbonyl, a thiocarbonyl, an imine, or an olefin, or R^(E2a) andR^(E2b) together form a carbonyl, a thiocarbonyl, an imine, or anolefin, or R^(E3a) and R^(E3b) together form a carbonyl, a thiocarbonyl,an imine, or an olefin, or R^(E4a) and R^(E4b) together form a carbonyl,a thiocarbonyl, an imine, or an olefin.

In some embodiments, a bond of the compound, such as the bond connectingE¹ and E², the bond connecting E² and E³, or the bond connecting E³ andE⁴, is fused to an additional ring system.

Non-limiting examples of a cyclic group, such as Cy¹ or Cy², includegroups of the following moieties, any of which is unsubstituted orsubstituted with any substituent described herein: cyclopropyl,cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl,cycloheptyl, cycloheptenyl, cyclooctyl, cyclooctenyl,cyclopenta-1,3-dienyl, cyclohexa-1,3-dienyl, cyclohexa-1,4-dienyl,cyclohepta-1,3-dienyl, cyclohepta-1,4-dienyl, bicyclo[3.2.0]heptanyl,octahydropentalenyl, octahydro-1H-indenyl, decahydroazulenyl,bicyclo[4.2.0]octanyl, decahydronaphthalenyl,decahydro-1H-benzo[7]annulenyl, dodecahydro-1H-fluorenyl,tetradecahydroanthracenyl, tetradecahydrophenanthrenyl,dodecahydros-indacenyl, dodecahydro-as-indacenyl,dodecahydro-1H-cyclopenta[b]napthalenyl,dodecahydro-1H-cyclopenta[a]napthalenyl, 1,2,3,4-tetrahydronaphthalenyl,4,5,6,7-tetrahydro-1H-indenyl, 2,3-dihydro-1H-indenyl,spiro[4.5]decanyl, spiro[5.5]undecanyl, spiro[4.4]nonanyl,spiro[2.5]octanyl, 9,10-dihydroanthracenyl,4,9-dihydro-1H-cyclopenta[b]napthalenyl, 9H-fluorenyl,(1Z,3Z,5Z)-cyclohepta-1,3,5-triene, benzimidazolyl, indolyl, indolinyl,indazolyl, isoxazolyl, 4-azaindolyl, 7-azaindolyl, imidazopyrimidinyl,benzofuranyl, benzothiophenyl, quinolinyl, isoquinolinyl, quinazolinyl,quinoxalinyl, purinyl, quinolizinyl, cinnolinyl, indolizinyl,phthalazinyl, isoindolyl, pteridinyl, benzofurazanyl, benzothiazolyl,benzoxazolyl, naphthyridinyl, furopyridinyl, benzoquinonyl,anthraquinonyl, 1,4-napthoquinonyl, acridinyl, azulenyl, indenyl,decalinyl, xanthenyl, 2H-chromenyl, dibenzofuranyl, dibenzopyrrolyl,phenoxazinyl, phenazinyl, phenoxathiinyl, phenyl, naphthalenyl,anthracenyl, phenanthrenyl, chrysenyl, pyrenyl, indanyl, tetralinyl,fluorenyl, acenaphthylenyl, acenaphthrene, fluoranthenyl, triphenylenyl,norbornanyl, 3-azabicyclo[3.1.0]hexanyl, 3-azabicyclo[4.1.0]heptanyl,1,4-dihydro-1,4-ethanoanthracenyl,1,4,5,8-tetrahydro-1,4,5,8-dimethanoanthracenyl, bicyclo[2.2.1]heptanyl,bicyclo[2.2.1]hept-2-enyl, bicyclo[2.2.2]octanyl,bicyclo[2.2.2]oct-2-enyl, bicyclo[2.2.1]heptanyl,bicyclo[3.1.1]heptanyl, bicyclo[4.4.0]decanyl, adamantanyl,quinuclidinyl, oxiranyl, oxetanyl, 2-tetrahydrofuranyl,3-tetrahydrofuranyl, 2-tetrahydropyranyl, 3-tetrahydropyranyl,4-tetrahydropyranyl, oxepanyl, oxocanyl, furanyl, 4H-pyranyl,(2Z,4Z,6Z)-oxepinyl, furfuralyl, dihydrofuranyl, dihydropyranyl,1,3-dioxolanyl, 1,4-dioxanyl, 1,3-dioxanyl, 1,2-dioxanyl, aziridinyl,azetidinyl, pyrrolidinyl, 1-pyrrolidinyl, 2-pyrrolidinyl,3-pyrrolidinyl, 1-piperidinyl, 2-piperidinyl, 3-piperidinyl,4-piperidinyl, azepanyl, azocanyl, piperidino, homopiperidinyl,2-pyrrolinyl, 3-pyrrolinyl, 2H-pyranyl, 4H-pyranyl, pyrazolidinyl,furazanyl, piperidinyl-N-oxide, morpholinyl-N-oxide,1-oxo-1-thiomorpholinyl, 1,1-dioxo-1-thiomorpholinyl, pyrrolyl,(2Z,4Z,6Z)-1H-azepinyl, imidazolyl, pyrazolyl, 1,2,3-triazolyl,1,2,4-triazolyl, 1,3,4-oxadiazolyl, 1,2,5-oxadiazolyl,1,3,4-thiadiazolyl, tetrazolyl, isothiazolyl, pyridinyl, pyrimidinyl,pyridazinyl, pyrazinyl, triazinyl, 1-piperazinyl, 2-piperazinyl,1,4-dihydropyrazinyl, 2-morpholinyl, 3-morpholinyl, 4-morpholinyl,morpholino, oxazolyl, thiazolyl, pyrrolidonyl, azetidinonyl,piperidinonyl, 4-thiazolidinyl, 2H-imidazol-2-one, phthalimidine,benzoxanyl, benzo[1,3]dioxinyl, benzo[1,4]dionyl, benzopyrrolidinyl,benzopiperidinl, benzoxolanyl, benzothiolanyl,4,5,6,7-tetrahydropyrazol[1,5-a]pyridinyl, benzothianyl, oxazepinyl,diazepinyl, thiazepinyl, 1,2,3,6-tetrahydropyridinyl, pyrazolinyl,imidazolinyl, imidazolidinyl, thiiranyl, thietanyl,2-tetrahydrothiophenyl, 3-tetrahydrothiophenyl, tetrahydrothiopyranyl,thiepanyl, thiocanyl, thiepinyl, thiophenyl, 4H-thiopyranyl,1,4-dithiinyl, 1,4-dithianyl, 2-thiomorpholinyl, 3-thiomorpholinyl,4-thiomorpholinyl, thiomorpholino, 1,3-dithiolanyl, dihydrothienyl,thienyl, silolyl, 3,4,5,6-tetrahydro-2H-azepinyl, 1,4-thiazepinyl,azocinyl, azonanyl, thioninyl, azecinyl, dihydrofuran-2(3H)-onyl,2,3-dioxolan-2-onyl, pyrrolidin-2-onyl, imidazolidin-2-onyl,piperidin-2-onyl, 1,3-oxazinan-2-onyl, phthalic anhydridyl, oxindolyl,indoline-2,3-dionyl, and 2,5-dihydrofuranyl.

Non-limiting examples of compounds of the invention include compounds ofthe following formula:

wherein: R¹ is hydrogen or an ester group; R² is hydrogen, halogen, orL¹-Ar¹; R³ is hydrogen, halogen, or L²-Ar²; or R² and R³ together withthe atoms to which R² and R³ are bound form a substituted orunsubstituted ring; each L¹ and L² is independently a linking group or abond; each Ar¹ is independently a substituted or unsubstituted arylgroup wherein Ar² is not substituted with an amide, amine, nitro, imine,or an ester; each Ar² is independently a substituted or unsubstitutedaryl group wherein Ar² is not substituted with an amide, amine, nitro,imine, or an ester; each A¹, A², A³, and A⁴ is independently C(R^(1a)),C(R^(1a))(R^(1b)), N, or N(R^(1a)); each R^(1a) and R^(1b) isindependently hydrogen, halogen, hydroxyl, sulfhydryl, nitro, nitroso,cyano, azido, a sulfoxide group, a sulfone group, a sulfonamide group, asulfonic acid group, an imine group, an acyl group, an acyloxy group,alkyl, alkenyl, alkynyl, an alkoxy group, an ether group, a carboxylicacid group, a carboxaldehyde group, an ester group, an amine group, anamide group, a carbonate group, a carbamate group, a thioether group, athioester group, a thioacid group, aryl, aryloxy, arylalkyl, arylalkoxy,heterocyclyl, heterocyclylalkyl, heteroaryl, or heteroarylalkyl, any ofwhich is substituted or unsubstituted; each

is independently a single or double bond, andpharmaceutically-acceptable salts thereof.

In some embodiments, when Ar¹ is phenyl brominated at one position, thenAr² is substituted on at least one position. In some embodiments, whenAr¹ is phenyl substituted with one methoxy group, then Ar² issubstituted on at least one position.

In some embodiments, when Ar¹ is substituted, Ar² is also substituted.In some embodiments, when Ar¹ is unsubstituted, Ar² is substituted.

In some embodiments, both L¹ and L² are independently

In some embodiments, both Ar¹ and Ar² are independently substituted withhydrogen, halogen, or aryloxy. In some embodiments, each linking groupis independently alkylene, alkenylene, O, S, SO₂, CO, N₂, or a bond.

In some embodiments, each

is independently chosen to provide an aromatic system.

In some embodiments, non-limiting examples of compounds of the inventioninclude compounds of the following formula:

wherein: R¹ is hydrogen or an ester group; R is hydrogen, halogen, orL¹-Ar¹; R³ is hydrogen, halogen, or L²-Ar²; or R² and R³ together withthe atoms to which R² and R³ are bound form a substituted orunsubstituted ring; each L¹ and L² is independently a linking group or abond; each Ar¹ is independently a substituted or unsubstituted arylgroup wherein Ar² is not substituted with an amide, amine, nitro, imine,or an ester; each Ar² is independently a substituted or unsubstitutedaryl group wherein Ar² is not substituted with an amide, amine, nitro,imine, or an ester; each A¹, A², A³, and A⁴ is independently C(R^(1a)),C(R^(1a))(R^(1b)), N, or N(R^(1a)); each R^(1a) and R^(1b) isindependently hydrogen, halogen, hydroxyl, sulfhydryl, nitro, nitroso,cyano, azido, a sulfoxide group, a sulfone group, a sulfonamide group, asulfonic acid group, an imine group, an acyl group, an acyloxy group,alkyl, alkenyl, alkynyl, an alkoxy group, an ether group, a carboxylicacid group, a carboxaldehyde group, an ester group, an amine group, anamide group, a carbonate group, a carbamate group, a thioether group, athioester group, a thioacid group, aryl, aryloxy, arylalkyl, arylalkoxy,heterocyclyl, heterocyclylalkyl, heteroaryl, or heteroarylalkyl, any ofwhich is substituted or unsubstituted; and pharmaceutically-acceptablesalts thereof.

In some embodiments, when Ar¹ is phenyl brominated at one position, thenAr² is substituted on at least one position. In some embodiments, whenAr¹ is phenyl substituted with one methoxy group, then Ar² issubstituted on at least one position.

In some embodiments, when Ar¹ is substituted, Ar² is also substituted.In some embodiments, when Ar¹ is unsubstituted, Ar² is substituted.

In some embodiments, both L¹ and L² are independently

In some embodiments, both Ar¹ and Ar² are independently substituted withhydrogen, halogen, or aryloxy. In some embodiments, each linking groupis independently alkylene, alkenylene, O, S, SO₂, CO, N₂, or a bond.

Non-limiting examples of compounds of the invention include thefollowing:

and pharmaceutically-acceptable salts thereof.

Any compound herein can be any or all stereoisomers, enantiomers,diastereomers, mixtures, racemates, atropisomers, and tautomers thereof.

A compound herein can bind a cellular target that is associated with adrug resistance mechanism, for example, an efflux pump. The binding cancause a decrease in the efficacy of the drug resistance mechanism,thereby increasing the efficacy of the compound within the cell. Acompound herein can cause a decrease in efficacy of a drug resistancemechanism that is, for example, about 2-fold, about 3-fold, about4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about9-fold, about 10-fold, about 11-fold, about 12-fold, about 13-fold,about 14-fold, about 15-fold, about 16-fold, about 17-fold, about18-fold, about 19-fold, about 20-fold, about 25-fold, about 30-fold,about 35-fold, about 40-fold, about 45-fold, about 50-fold, about55-fold, about 60-fold, about 65-fold, about 70-fold, about 75-fold,about 80-fold, about 85-fold, about 90-fold, about 95-fold, about100-fold, about 110-fold, about 120-fold, about 130-fold, about140-fold, about 150-fold, about 160-fold, about 170-fold, about180-fold, about 190-fold, about 200-fold, about 250-fold, about300-fold, about 350-fold, about 400-fold, about 450-fold, about500-fold, about 550-fold, about 600-fold, about 650-fold, about700-fold, about 750-fold, about 800-fold, about 850-fold, about900-fold, about 950-fold, about 1000-fold, about 1500-fold, or about2000-fold in comparison to the efficacy of the drug resistance mechanismin a cell that has not been treated with the compound.

Optional Substituents for Chemical Groups.

Non-limiting examples of optional substituents include hydroxyl groups,sulfhydryl groups, halogens, amino groups, nitro groups, nitroso groups,cyano groups, azido groups, sulfoxide groups, sulfone groups,sulfonamide groups, carboxyl groups, carboxaldehyde groups, iminegroups, alkyl groups, halo-alkyl groups, alkenyl groups, halo-alkenylgroups, alkynyl groups, halo-alkynyl groups, alkoxy groups, aryl groups,aryloxy groups, aralkyl groups, arylalkoxy groups, heterocyclyl groups,acyl groups, acyloxy groups, carbamate groups, amide groups, urethanegroups, and ester groups.

Non-limiting examples of alkyl and alkylene groups include straight,branched, and cyclic alkyl and alkylene groups. An alkyl or alkylenegroup can be, for example, a C₁, C₂, C₃, C₄, C₅, C₆, C₇, C₈, C₉, C₁₀,C₁₁, C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, C₂₀, C₂₁, C₂₂, C₂₃, C₂₄,C₂₅, C₂₆, C₂₇, C₂₈, C₂₉, C₃₀, C₃₁, C₃₂, C₃₃, C₃₄, C₃₅, C₃₆, C₃₇, C₃₈,C₃₉, C₄₀, C₄₁, C₄₂, C₄₃, C₄₄, C₄₅, C₄₆, C₄₇, C₄₈, C₄₉, or C₅₀ group thatis substituted or unsubstituted.

Non-limiting examples of straight alkyl groups include methyl, ethyl,propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, and decyl.

Branched alkyl groups include any straight alkyl group substituted withany number of alkyl groups. Non-limiting examples of branched alkylgroups include isopropyl, isobutyl, sec-butyl, and t-butyl.

Non-limiting examples of cyclic alkyl groups include cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptlyl, and cyclooctylgroups. Cyclic alkyl groups also include fused-, bridged-, andspiro-bicycles and higher fused-, bridged-, and spiro-systems. A cyclicalkyl group can be substituted with any number of straight, branched, orcyclic alkyl groups.

Non-limiting examples of alkenyl and alkenylene groups include straight,branched, and cyclic alkenyl groups. The olefin or olefins of an alkenylgroup can be, for example, E, Z, cis, trans, terminal, or exo-methylene.An alkenyl or alkenylene group can be, for example, a C₂, C₃, C₄, C₅,C₆, C₇, C₈, C₉, C₁₀, C₁₁, C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, C₂₀,C₂₁, C₂₂, C₂₃, C₂₄, C₂₅, C₂₆, C₂₇, C₂₈, C₂₉, C₃₀, C₃₁, C₃₂, C₃₃, C₃₄,C₃₅, C₃₆, C₃₇, C₃₈, C₃₉, C₄₀, C₄₁, C₄₂, C₄₃, C₄₄, C₄₅, C₄₆, C₄₇, C₄₈,C₄₉, or C₅₀ group that is substituted or unsubstituted.

Non-limiting examples of alkynyl or alkynylene groups include straight,branched, and cyclic alkynyl groups. The triple bond of an alkylnyl oralkynylene group can be internal or terminal. An alkylnyl or alkynylenegroup can be, for example, a C₂, C₃, C₄, C₅, C₆, C₇, C₈, C₉, C₁₀, C₁₁,C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, C₂₀, C₂₁, C₂₂, C₂₃, C₂₄, C₂₅,C₂₆, C₂₇, C₂₈, C₂₉, C₃₀, C₃₁, C₃₂, C₃₃, C₃₄, C₃₅, C₃₆, C₃₇, C₃₈, C₃₉,C₄₀, C₄₁, C₄₂, C₄₃, C₄₄, C₄₅, C₄₆, C₄₇, C₄₈, C₄₉, or C₅₀ group that issubstituted or unsubstituted.

A halo-alkyl group can be any alkyl group substituted with any number ofhalogen atoms, for example, fluorine, chlorine, bromine, and iodineatoms. A halo-alkenyl group can be any alkenyl group substituted withany number of halogen atoms. A halo-alkynyl group can be any alkynylgroup substituted with any number of halogen atoms.

An alkoxy group can be, for example, an oxygen atom substituted with anyalkyl, alkenyl, or alkynyl group. An ether or an ether group comprisesan alkoxy group. Non-limiting examples of alkoxy groups include methoxy,ethoxy, propoxy, isopropoxy, and isobutoxy.

An aryl group can be heterocyclic or non-heterocyclic. An aryl group canbe monocyclic or polycyclic. An aryl group can be substituted with anynumber of substituents described herein, for example, hydrocarbylgroups, alkyl groups, alkoxy groups, and halogen atoms. Non-limitingexamples of aryl groups include phenyl, toluyl, naphthyl, pyrrolyl,pyridyl, imidazolyl, thiophenyl, and furyl.

An aryloxy group can be, for example, an oxygen atom substituted withany aryl group, such as phenoxy.

An aralkyl group can be, for example, any alkyl group substituted withany aryl group, such as benzyl.

An arylalkoxy group can be, for example, an oxygen atom substituted withany aralkyl group, such as benzyloxy.

A heterocycle can be any ring containing a ring atom that is not carbon,for example, N, O, S, P, Si, B, or any other heteroatom. A heterocyclecan be substituted with any number of substituents, for example, alkylgroups and halogen atoms. A heterocycle can be aromatic (heteroaryl) ornon-aromatic. Non-limiting examples of heterocycles include pyrrole,pyrrolidine, pyridine, piperidine, succinamide, maleimide, morpholine,imidazole, thiophene, furan, tetrahydrofuran, pyran, andtetrahydropyran.

An acyl group can be, for example, a carbonyl group substituted withhydrocarbyl, alkyl, hydrocarbyloxy, alkoxy, aryl, aryloxy, aralkyl,arylalkoxy, or a heterocycle. Non-limiting examples of acyl includeacetyl, benzoyl, benzyloxycarbonyl, phenoxycarbonyl, methoxycarbonyl,and ethoxycarbonyl.

An acyloxy group can be an oxygen atom substituted with an acyl group.An ester or an ester group comprises an acyloxy group. A non-limitingexample of an acyloxy group, or an ester group, is acetate.

A carbamate group can be an oxygen atom substituted with a carbamoylgroup, wherein the nitrogen atom of the carbamoyl group isunsubstituted, monosubstituted, or disubstituted with one or more ofhydrocarbyl, alkyl, aryl, heterocyclyl, or aralkyl. When the nitrogenatom is disubstituted, the two substituents together with the nitrogenatom can form a heterocycle.

Pharmaceutically-Acceptable Salts.

The invention provides the use of pharmaceutically-acceptable salts ofany therapeutic compound described herein. Pharmaceutically-acceptablesalts include, for example, acid-addition salts and base-addition salts.The acid that is added to the compound to form an acid-addition salt canbe an organic acid or an inorganic acid. A base that is added to thecompound to form a base-addition salt can be an organic base or aninorganic base. In some embodiments, a pharmaceutically-acceptable saltis a metal salt. In some embodiments, a pharmaceutically-acceptable saltis an ammonium salt.

Metal salts can arise from the addition of an inorganic base to acompound of the invention. The inorganic base consists of a metal cationpaired with a basic counterion, such as, for example, hydroxide,carbonate, bicarbonate, or phosphate. The metal can be an alkali metal,alkaline earth metal, transition metal, or main group metal. In someembodiments, the metal is lithium, sodium, potassium, cesium, cerium,magnesium, manganese, iron, calcium, strontium, cobalt, titanium,aluminum, copper, cadmium, or zinc.

In some embodiments, a metal salt is a lithium salt, a sodium salt, apotassium salt, a cesium salt, a cerium salt, a magnesium salt, amanganese salt, an iron salt, a calcium salt, a strontium salt, a cobaltsalt, a titanium salt, an aluminum salt, a copper salt, a cadmium salt,or a zinc salt.

Ammonium salts can arise from the addition of ammonia or an organicamine to a compound of the invention. In some embodiments, the organicamine is triethyl amine, diisopropyl amine, ethanol amine, diethanolamine, triethanol amine, morpholine, N-methylmorpholine, piperidine,N-methylpiperidine, N-ethylpiperidine, dibenzylamine, piperazine,pyridine, pyrrazole, pipyrrazole, imidazole, pyrazine, or pipyrazine.

In some embodiments, an ammonium salt is a triethyl amine salt, adiisopropyl amine salt, an ethanol amine salt, a diethanol amine salt, atriethanol amine salt, a morpholine salt, an N-methylmorpholine salt, apiperidine salt, an N-methylpiperidine salt, an N-ethylpiperidine salt,a dibenzylamine salt, a piperazine salt, a pyridine salt, a pyrrazolesalt, a pipyrrazole salt, an imidazole salt, a pyrazine salt, or apipyrazine salt.

Acid addition salts can arise from the addition of an acid to a compoundof the invention. In some embodiments, the acid is organic. In someembodiments, the acid is inorganic. In some embodiments, the acid ishydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid,nitrous acid, sulfuric acid, sulfurous acid, a phosphoric acid,isonicotinic acid, lactic acid, salicylic acid, tartaric acid, ascorbicacid, gentisinic acid, gluconic acid, glucaronic acid, saccaric acid,formic acid, benzoic acid, glutamic acid, pantothenic acid, acetic acid,propionic acid, butyric acid, fumaric acid, succinic acid,methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid,p-toluenesulfonic acid, citric acid, oxalic acid, or maleic acid.

In some embodiments, the salt is a hydrochloride salt, a hydrobromidesalt, a hydroiodide salt, a nitrate salt, a nitrite salt, a sulfatesalt, a sulfite salt, a phosphate salt, isonicotinate salt, a lactatesalt, a salicylate salt, a tartrate salt, an ascorbate salt, agentisinate salt, a gluconate salt, a glucaronate salt, a saccaratesalt, a formate salt, a benzoate salt, a glutamate salt, a pantothenatesalt, an acetate salt, a propionate salt, a butyrate salt, a fumaratesalt, a succinate salt, a methanesulfonate (mesylate) salt, anethanesulfonate salt, a benzenesulfonate salt, a p-toluenesulfonatesalt, a citrate salt, an oxalate salt, or a maleate salt.

Purity of Compounds of the Invention.

Any compound herein can be purified. A compound herein can be least 1%pure, at least 2% pure, at least 3% pure, at least 4% pure, at least 5%pure, at least 6% pure, at least 7% pure, at least 8% pure, at least 9%pure, at least 10% pure, at least 11% pure, at least 12% pure, at least13% pure, at least 14% pure, at least 15% pure, at least 16% pure, atleast 17% pure, at least 18% pure, at least 19% pure, at least 20% pure,at least 21% pure, at least 22% pure, at least 23% pure, at least 24%pure, at least 25% pure, at least 26% pure, at least 27% pure, at least28% pure, at least 29% pure, at least 30% pure, at least 31% pure, atleast 32% pure, at least 33% pure, at least 34% pure, at least 35% pure,at least 36% pure, at least 37% pure, at least 38% pure, at least 39%pure, at least 40% pure, at least 41% pure, at least 42% pure, at least43% pure, at least 44% pure, at least 45% pure, at least 46% pure, atleast 47% pure, at least 48% pure, at least 49% pure, at least 50% pure,at least 51% pure, at least 52% pure, at least 53% pure, at least 54%pure, at least 55% pure, at least 56% pure, at least 57% pure, at least58% pure, at least 59% pure, at least 60% pure, at least 61% pure, atleast 62% pure, at least 63% pure, at least 64% pure, at least 65% pure,at least 66% pure, at least 67% pure, at least 68% pure, at least 69%pure, at least 70% pure, at least 71% pure, at least 72% pure, at least73% pure, at least 74% pure, at least 75% pure, at least 76% pure, atleast 77% pure, at least 78% pure, at least 79% pure, at least 80% pure,at least 81% pure, at least 82% pure, at least 83% pure, at least 84%pure, at least 85% pure, at least 86% pure, at least 87% pure, at least88% pure, at least 89% pure, at least 90% pure, at least 91% pure, atleast 92% pure, at least 93% pure, at least 94% pure, at least 95% pure,at least 96% pure, at least 97% pure, at least 98% pure, at least 99%pure, at least 99.1% pure, at least 99.2% pure, at least 99.3% pure, atleast 99.4% pure, at least 99.5% pure, at least 99.6% pure, at least99.7% pure, at least 99.8% pure, or at least 99.9% pure.

Light-Activation of Compounds of the Invention.

Compounds disclosed herein can be effective as efflux pump inhibitors(EPIs). The compounds can inhibit efflux pumps by direct binding. Thebinding of the efflux pumps by the compounds can prevent expulsion ofantibiotics that have been administered for treatment of microbialinfections. Compounds of the invention can compete with commonly-usedEPIs.

The present disclosure describes compounds that can act as bacterialefflux inhibitors. Non-limiting examples of illustrative compounds canbe based on the 2,3-di-((E)-2-arylethenyl)indole structural scaffold.

Inactive 2,3-di-((E)-2-arylethenyl)indoles, which have low bactericidalactivity unless used in combination with known antibiotics, can beconverted to potent antibacterial agents as single agents vialight-activation. Low doses of compounds of the invention irradiatedwith white light for 2 minutes can kill Gram-positive organismsincluding, for example, hospital-acquired MRSA, community-acquired MRSA,Staphylococcus aureus, vancomycin-resistant Enterococcus (VRE),Streptococcus pyogenes, and Streptococcus mutans (Ward's 85W).Gram-negative bacteria, for example, Acinetobacter baumannii,Escherichia coli, Klebsiella pneumoniae, and Carbapenem-resistantenterobacteriaceae, can also be susceptible to treatment with compoundsof the invention in the presence of non-toxic doses of PMB.Light-induced killing of bacteria with 2,3-di-((E)-2-arylethenyl)indolescan represent a therapeutic strategy in the treatment of localizedinfections involving resistant microorganisms.

In some embodiments, compounds of the invention can be photoactive,photosensitive, photodynamic, or photoresponsive. The compound can beused for photodynamic therapy, wherein the compound can be aphotosensitizer and lead to the generation of, for example, singletoxygen and reactive oxygen species (ROS).

Wavelengths of light that can be used in a method of the inventioninclude, for example, about 200 nm, about 210 nm, about 220 nm, about230 nm, about 240 nm, about 250 nm, about 260 nm, about 270 nm, about280 nm, about 290 nm, about 300 nm, about 310 nm, about 320 nm, about330 nm, about 340 nm, about 350 nm, about 360 nm, about 370 nm, about380 nm, about 390 nm, about 400 nm, about 410 nm, about 420 nm, about430 nm, about 440 nm, about 450 nm, about 460 nm, about 470 nm, about480 nm, about 490 nm, about 500 nm, about 510 nm, about 520 nm, about530 nm, about 540 nm, about 550 nm, about 560 nm, about 570 nm, about580 nm, about 590 nm, about 600 nm, about 610 nm, about 620 nm, about630 nm, about 640 nm, about 650 nm, about 660 nm, about 670 nm, about680 nm, about 690 nm, about 700 nm, about 710 nm, about 720 nm, about730 nm, about 740 nm, about 750 nm, about 760 nm, about 770 nm, about780 nm, about 790 nm, and about 800 nm.

In some embodiments, compounds of the invention can be applied topicallyto the skin, or a body cavity, for example, oral, vaginal, bladder,cranial, spinal, thoracic, or pelvic cavity of a subject. In someembodiments, the compounds of the invention can be applied to anaccessible body cavity. The compound can be then be activated viaexposure of the skin to natural or artificial light. The skin can beexposed to light, for example, for about 1 second, about 2 seconds,about 3 seconds, about 4 seconds, about 5 seconds, about 6 seconds,about 7 seconds, about 8 seconds, about 9 seconds, about 10 seconds,about 15 seconds, about 20 seconds, about 25 seconds, about 30 seconds,about 35 seconds, about 40 seconds, about 45 seconds, about 50 seconds,about 55 seconds, about 1 minute, about 1.5 minutes, about 2 minutes,about 2.5 minutes, about 3 minutes, about 3.5 minutes, about 4 minutes,about 4.5 minutes, about 5 minutes, about 6 minutes, about 7 minutes,about 8 minutes, about 9 minutes, about 10 minutes, about 15 minutes, orabout 20 minutes.

The depth of administration of the compounds in the skin can be, forexample, about 0.1 mm, about 0.2 mm, about 0.3 mm, about 0.4 mm, about0.5 mm, about 0.6 mm, about 0.7 mm, about 0.8 mm, about 0.9 mm, about 1mm, about 1.1 mm, about 1.2 mm, about 1.3 mm, about 1.4 mm, about 1.5mm, about 1.6 mm, about 1.7 mm, about 1.8 mm, about 1.9 mm, about 2 mm,about 2.1 mm, about 2.2 mm, about 2.3 mm, about 2.4 mm, about 2.5 mm,about 2.6 mm, about 2.7 mm, about 2.8 mm, about 2.9 mm, about 3 mm,about 3.1 mm, about 3.2 mm, about 3.3 mm, about 3.4 mm, about 3.5 mm,about 3.6 mm, about 3.7 mm, about 3.8 mm, about 3.9 mm, about 4 mm,about 4.1 mm, about 4.2 mm, about 4.3 mm, about 4.4 mm, about 4.5 mm,about 4.6 mm, about 4.7 mm, about 4.8 mm, about 4.9 mm, and about 5 mm.

Activation of the compound can occur via exposure to light wherein theadministration of the light is continuous or pulsed. Pulses of light canbe separated by, for example, about 1 second, about 2 seconds, about 3seconds, about 4 seconds, about 5 seconds, about 6 seconds, about 7seconds, about 8 seconds, about 9 seconds, about 10 seconds, about 15seconds, about 20 seconds, about 25 seconds, about 30 seconds, about 35seconds, about 40 seconds, about 45 seconds, about 50 seconds, about 55seconds, about 1 minute, about 1.5 minutes, about 2 minutes, about 2.5minutes, about 3 minutes, about 3.5 minutes, about 4 minutes, about 4.5minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8minutes, about 9 minutes, about 10 minutes, about 15 minutes, or about20 minutes.

In some embodiments, activation of the compound via light can occurconcurrently with, or subsequent to, administration of the compound to asubject. Light can then be administered to the subject, for example,every 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 25minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8hours, 9 hours, 10 hours, 11 hours, 12 hours, 18 hours, 1 day, 2 days, 3days, 4 days, 5 days, 6 days, or 1 week to improve efficacy of thecompound.

The energy of light used to activate the compound can be, for example,about 10 J/cm², about 15 J/cm², about 20 J/cm², about 25 J/cm², about 30J/cm², about 35 J/cm², about 40 J/cm², about 45 J/cm², about 50 J/cm²,about 55 J/cm², about 60 J/cm², about 65 J/cm², about 66 J/cm², about 67J/cm², about 68 J/cm², about 69 J/cm², about 70 J/cm², about 71 J/cm²,about 72 J/cm², about 73 J/cm², about 74 J/cm², about 75 J/cm², about 76J/cm², about 77 J/cm², about 78 J/cm², about 79 J/cm², about 80 J/cm²,about 81 J/cm², about 82 J/cm², about 83 J/cm², about 84 J/cm², about 85J/cm², about 86 J/cm², about 87 J/cm², about 88 J/cm², about 89 J/cm²,about 90 J/cm², about 95 J/cm², or about 100 J/cm².

The brightness of light used to activate the compound can be, forexample, about 100 lm, about 110 lm, about 120 lm, about 130 lm, about140 lm, about 150 lm, about 160 lm, about 170 lm, about 180 lm, about190 lm, about 200 lm, about 250 lm, about 300 lm, about 350 lm, about450 lm, about 500 lm, about 550 lm, about 600 lm, about 650 lm, about700 lm, about 750 lm, about 800 lm, about 850 lm, about 900 lm, about950 lm, about 1000 lm, about 1100 lm, about 1200 lm, about 1300 lm,about 1400 lm, about 1500 lm, about 1600 lm, about 1700 lm, about 1800lm, about 1900 lm, about 2000 lm, about 2500 lm, about 3000 lm, about3500 lm, or about 4000 lm.

When exposed to continuous light, compounds of the invention can have anincrease in activity that is, for example, about 2-fold, about 3-fold,about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold,about 9-fold, about 10-fold, about 11-fold, about 12-fold, about13-fold, about 14-fold, about 15-fold, about 16-fold, about 17-fold,about 18-fold, about 19-fold, about 20-fold, about 25-fold, about30-fold, about 35-fold, about 40-fold, about 45-fold, about 50-fold,about 55-fold, about 60-fold, about 65-fold, about 70-fold, about75-fold, about 80-fold, about 85-fold, about 90-fold, about 95-fold,about 100-fold, about 110-fold, about 120-fold, about 130-fold, about140-fold, about 150-fold, about 160-fold, about 170-fold, about180-fold, about 190-fold, about 200-fold, about 250-fold, about300-fold, about 350-fold, about 400-fold, about 450-fold, about500-fold, about 550-fold, about 600-fold, about 650-fold, about700-fold, about 750-fold, about 800-fold, about 850-fold, about900-fold, about 950-fold, about 1000-fold, about 1500-fold, or about2000-fold greater than when the compound is not exposed to continuouslight.

When exposed to pulsed light, compounds of the invention can have anincrease in activity that is, for example, about 2-fold, about 3-fold,about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold,about 9-fold, about 10-fold, about 11-fold, about 12-fold, about13-fold, about 14-fold, about 15-fold, about 16-fold, about 17-fold,about 18-fold, about 19-fold, about 20-fold, about 25-fold, about30-fold, about 35-fold, about 40-fold, about 45-fold, about 50-fold,about 55-fold, about 60-fold, about 65-fold, about 70-fold, about75-fold, about 80-fold, about 85-fold, about 90-fold, about 95-fold,about 100-fold, about 110-fold, about 120-fold, about 130-fold, about140-fold, about 150-fold, about 160-fold, about 170-fold, about180-fold, about 190-fold, about 200-fold, about 250-fold, about300-fold, about 350-fold, about 400-fold, about 450-fold, about500-fold, about 550-fold, about 600-fold, about 650-fold, about700-fold, about 750-fold, about 800-fold, about 850-fold, about900-fold, about 950-fold, about 1000-fold, about 1500-fold, or about2000-fold greater than when the compound is not exposed to constantlight.

Bacterial strains that can be treated by a method of the invention canbe gram-negative or gram-positive. Non-limiting examples of microbesthat can be treated by a method of the invention include Acinetobacterbaumannii, carbapenem-resistant Enterobacteriaceae (CRE),clindamycin-resistant Group B Streptococcus, Clostridium difficile,drug-resistant Campylobacter, drug-resistant Neisseria gonorrhoeae,drug-resistant non-typhoidal Salmonella, drug-resistant Salmonellatyphi, drug-resistant Shigella, drug-resistant Streptococcus pneumoniae,drug-resistant tuberculosis, erythromycin-resistant Group AStreptococcus, Escherichia coli, extended spectrum β-lactamase producingEnterobacteriaceae (ESBLs), fluconazole-resistant Candida,methicillin-resistant S. aureus (MRSA), multidrug-resistantAcinetobacter, multidrug-resistant Pseudomonas aeruginosa, S. aureus,VRE, and vancomycin-resistant S. aureus (VRSA). In some embodiments, themethods of the invention can be applied to agricultural pathogens.

In some embodiments, a therapy of the disclosure has synergisticactivity in combination with an antibiotic. Synergy can refer to theobservation that the combination of two therapeutic agents can have anoverall effect that is greater than the sum of the two individualeffects. Synergy can also refer to the observation that a single drugproduces no effect but, when administered with a second drug produces aneffect that is greater than the effect produced by the secondtherapeutic agent alone.

Classes of antibiotics that can be used in a method of inventioninclude, for example, aminoglycosides, ansamycins, β-lactams,carbapenems, cephalosporins, glycopeptides, lincosamides, lipopeptides,macrolides, monobactams, nitrofurans, oxazolidinones, penicillins,polypeptides, quinolones, fluroquinolones, sulfonamides, andtetracyclines.

Non-limiting examples of antibiotics that can be used in a method of theinvention include ampicillin, amoxicillin, azithromycin, carbenicillin,clarithromycin, dicloxicillin, doxycycline, erythromycin, gentamicin,kanamycin, methicillin, neomycin, norfloxacin, oxacillin, PMB,colisitin, penicillin, penicillin G, penicillin V, streptomycin,tetracycline, tobramycin, polyethyleneimine, lactic acid, benzoic acidbacitracin, imipenem, and vancomycin.

In some embodiments, compounds of the invention can be used to treat acondition caused by a microbe in a subject. In some embodiments, themicrobe can be a bacterium, fungus, or protozoa.

In some embodiments, compounds of the invention can be used to treatcancer in a subject. A compound of the invention can, for example, slowthe proliferation of cancer cell lines, or kill cancer cells.Non-limiting examples of cancer that can be treated by a compound of theinvention include: acute lymphoblastic leukemia, acute myeloid leukemia,adrenocortical carcinoma, AIDS-related cancers, AIDS-related lymphoma,anal cancer, appendix cancer, astrocytomas, basal cell carcinoma, bileduct cancer, bladder cancer, bone cancers, brain tumors, such ascerebellar astrocytoma, cerebral astrocytoma/malignant glioma,ependymoma, medulloblastoma, supratentorial primitive neuroectodermaltumors, visual pathway and hypothalamic glioma, breast cancer, bronchialadenomas, Burkitt lymphoma, carcinoma of unknown primary origin, centralnervous system lymphoma, cerebellar astrocytoma, cervical cancer,childhood cancers, chronic lymphocytic leukemia, chronic myelogenousleukemia, chronic myeloproliferative disorders, colon cancer, cutaneousT-cell lymphoma, desmoplastic small round cell tumor, endometrialcancer, ependymoma, esophageal cancer, Ewing's sarcoma, germ celltumors, gallbladder cancer, gastric cancer, gastrointestinal carcinoidtumor, gastrointestinal stromal tumor, gliomas, hairy cell leukemia,head and neck cancer, heart cancer, hepatocellular (liver) cancer,Hodgkin lymphoma, Hypopharyngeal cancer, intraocular melanoma, isletcell carcinoma, Kaposi sarcoma, kidney cancer, laryngeal cancer, lip andoral cavity cancer, liposarcoma, liver cancer, lung cancers, such asnon-small cell and small cell lung cancer, lymphomas, leukemias,macroglobulinemia, malignant fibrous histiocytoma of bone/osteosarcoma,medulloblastoma, melanomas, mesothelioma, metastatic squamous neckcancer with occult primary, mouth cancer, multiple endocrine neoplasiasyndrome, myelodysplastic syndromes, myeloid leukemia, nasal cavity andparanasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma,non-Hodgkin lymphoma, non-small cell lung cancer, oral cancer,oropharyngeal cancer, osteosarcoma/malignant fibrous histiocytoma ofbone, ovarian cancer, ovarian epithelial cancer, ovarian germ celltumor, pancreatic cancer, pancreatic cancer islet cell, paranasal sinusand nasal cavity cancer, parathyroid cancer, penile cancer, pharyngealcancer, pheochromocytoma, pineal astrocytoma, pineal germinoma,pituitary adenoma, pleuropulmonary blastoma, plasma cell neoplasia,primary central nervous system lymphoma, prostate cancer, rectal cancer,renal cell carcinoma, renal pelvis and ureter transitional cell cancer,retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcomas, skincancers, skin carcinoma merkel cell, small intestine cancer, soft tissuesarcoma, squamous cell carcinoma, stomach cancer, T-cell lymphoma,throat cancer, thymoma, thymic carcinoma, thyroid cancer, trophoblastictumor (gestational), cancers of unknown primary site, urethral cancer,uterine sarcoma, vaginal cancer, vulvar cancer, Waldenströmmacroglobulinemia, and Wilms tumor.

Subjects can be, for example, elderly adults, adults, adolescents,pre-adolescents, children, toddlers, infants, and non-human animals. Insome embodiments, a subject is a patient. Non-human animal subjects canbe, for example, a mouse, rat, a chicken, a rabbit, a dog, a cat, or acow. Compounds of the invention can be employed in places where thespread of drug-resistant bacteria can be more likely, for example,hospitals, nursing homes, dormitories, homeless shelters, militarybarracks, schools, locker rooms, gymnasiums, and prisons. The methods ofthe invention can be applied to, for example, fomites, surgicalinstruments, tables, chairs, doors, eating utensils, bedding, beds, andkeyboards.

In some embodiments, the methods of the invention can be applied to, forexample, a plant, a fungus, or a parasite. Administration can, forexample, kill or inhibit the Plant, fungus, or parasite, or kill orinhibit an agent that harms or presents a risk of harm to a plant orfungus, or lessen a likelihood of such risk. For example, agriculturalapplications to inhibit the spread of and damage byagriculturally-detrimental microbes are possible.

Pharmaceutical Compositions of the Invention.

A pharmaceutical composition of the invention can be used, for example,before, during, or after treatment of a subject with light, antibiotics,or another pharmaceutical agent.

A pharmaceutical composition of the invention can be a combination ofany pharmaceutical compounds described herein with other chemicalcomponents, such as carriers, stabilizers, diluents, dispersing agents,suspending agents, thickening agents, and/or excipients. Thepharmaceutical composition facilitates administration of the compound toan organism. Pharmaceutical compositions can be administered intherapeutically-effective amounts as pharmaceutical compositions byvarious forms and routes including, for example, intravenous,subcutaneous, intramuscular, oral, parenteral, ophthalmic, subcutaneous,transdermal, nasal, vaginal, and topical administration.

A pharmaceutical composition can be administered in a local manner, forexample, via injection of the compound directly into an organ,optionally in a depot or sustained release formulation or implant.Pharmaceutical compositions can be provided in the form of a rapidrelease formulation, in the form of an extended release formulation, orin the form of an intermediate release formulation. A rapid release formcan provide an immediate release. An extended release formulation canprovide a controlled release or a sustained delayed release.

For oral administration, pharmaceutical compositions can be formulatedby combining the active compounds with pharmaceutically-acceptablecarriers or excipients. Such carriers can be used to formulate liquids,gels, syrups, elixirs, slurries, or suspensions, for oral ingestion by asubject. Non-limiting examples of solvents used in an oral dissolvableformulation can include water, ethanol, isopropanol, saline,physiological saline, DMSO, dimethylformamide, potassium phosphatebuffer, phosphate buffer saline (PBS), sodium phosphate buffer,4-2-hydroxyethyl-1-piperazineethanesulfonic acid buffer (HEPES),3-(N-morpholino)propanesulfonic acid buffer (MOPS),piperazine-N,N′-bis(2-ethanesulfonic acid) buffer (PIPES), and salinesodium citrate buffer (SSC). Non-limiting examples of co-solvents usedin an oral dissolvable formulation can include sucrose, urea, cremaphor,DMSO, and potassium phosphate buffer.

Pharmaceutical preparations can be formulated for intravenousadministration. The pharmaceutical compositions can be in a formsuitable for parenteral injection as a sterile suspension, solution oremulsion in oily or aqueous vehicles, and can contain formulatory agentssuch as suspending, stabilizing and/or dispersing agents. Pharmaceuticalformulations for parenteral administration include aqueous solutions ofthe active compounds in water-soluble form. Suspensions of the activecompounds can be prepared as oily injection suspensions. Suitablelipophilic solvents or vehicles include fatty oils such as sesame oil,or synthetic fatty acid esters, such as ethyl oleate or triglycerides,or liposomes. The suspension can also contain suitable stabilizers oragents which increase the solubility of the compounds to allow for thepreparation of highly concentrated solutions. Alternatively, the activeingredient can be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

The active compounds can be administered topically and can be formulatedinto a variety of topically administrable compositions, such assolutions, suspensions, lotions, gels, pastes, medicated sticks, balms,creams, and ointments. Such pharmaceutical compositions can containsolubilizers, stabilizers, tonicity enhancing agents, buffers andpreservatives.

The compounds can also be formulated in rectal compositions such asenemas, rectal gels, rectal foams, rectal aerosols, suppositories, jellysuppositories, or retention enemas, containing conventional suppositorybases such as cocoa butter or other glycerides, as well as syntheticpolymers such as polyvinylpyrrolidone, and PEG. In suppository forms ofthe compositions, a low-melting wax such as a mixture of fatty acidglycerides, optionally in combination with cocoa butter, can be melted.

In practicing the methods of treatment or use provided herein,therapeutically-effective amounts of the compounds described herein areadministered in pharmaceutical compositions to a subject having adisease or condition to be treated. In some embodiments, the subject isa mammal such as a human. A therapeutically-effective amount can varywidely depending on the severity of the disease, the age and relativehealth of the subject, the potency of the compounds used, and otherfactors. The compounds can be used singly or in combination with one ormore therapeutic agents as components of mixtures.

Pharmaceutical compositions can be formulated using one or morephysiologically-acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the active compounds intopreparations that can be used pharmaceutically. Formulation can bemodified depending upon the route of administration chosen.Pharmaceutical compositions comprising a compound described herein canbe manufactured, for example, by mixing, dissolving, emulsifying,encapsulating, entrapping, or compression processes.

The pharmaceutical compositions can include at least onepharmaceutically-acceptable carrier, diluent, or excipient and compoundsdescribed herein as free-base or pharmaceutically-acceptable salt form.Pharmaceutical compositions can contain solubilizers, stabilizers,tonicity enhancing agents, buffers and preservatives.

Methods for the preparation of compositions comprising the compoundsdescribed herein include formulating the compounds with one or moreinert, pharmaceutically-acceptable excipients or carriers to form asolid, semi-solid, or liquid composition. Solid compositions include,for example, powders, tablets, dispersible granules, capsules, andcachets. Liquid compositions include, for example, solutions in which acompound is dissolved, emulsions comprising a compound, or a solutioncontaining liposomes, micelles, or nanoparticles comprising a compoundas disclosed herein. Semi-solid compositions include, for example, gels,suspensions and creams. The compositions can be in liquid solutions orsuspensions, solid forms suitable for solution or suspension in a liquidprior to use, or as emulsions. These compositions can also contain minoramounts of nontoxic, auxiliary substances, such as wetting oremulsifying agents, pH buffering agents, and otherpharmaceutically-acceptable additives.

Non-limiting examples of dosage forms suitable for use in the inventioninclude liquid, powder, gel, nanosuspension, nanoparticle, microgel,aqueous or oily suspensions, emulsion, and any combination thereof.

Non-limiting examples of pharmaceutically-acceptable excipients suitablefor use in the invention include binding agents, disintegrating agents,anti-adherents, anti-static agents, surfactants, anti-oxidants, coatingagents, coloring agents, plasticizers, preservatives, suspending agents,emulsifying agents, anti-microbial agents, spheronization agents, andany combination thereof.

A composition of the invention can be, for example, an immediate releaseform or a controlled release formulation. An immediate releaseformulation can be formulated to allow the compounds to act rapidly.Non-limiting examples of immediate release formulations include readilydissolvable formulations. A controlled release formulation can be apharmaceutical formulation that has been adapted such that release ratesand release profiles of the active agent can be matched to physiologicaland chronotherapeutic requirements or, alternatively, has beenformulated to effect release of an active agent at a programmed rate.Non-limiting examples of controlled release formulations includegranules, delayed release granules, hydrogels (e.g., of synthetic ornatural origin), other gelling agents (e.g., gel-forming dietaryfibers), matrix-based formulations (e.g., formulations comprising apolymeric material having at least one active ingredient dispersedthrough), granules within a matrix, polymeric mixtures, and granularmasses.

In some, a controlled release formulation is a delayed release form. Adelayed release form can be formulated to delay a compound's action foran extended period of time. A delayed release form can be formulated todelay the release of an effective dose of one or more compounds, forexample, for about 4, about 8, about 12, about 16, or about 24 hours.

A controlled release formulation can be a sustained release form. Asustained release form can be formulated to sustain, for example, thecompound's action over an extended period of time. A sustained releaseform can be formulated to provide an effective dose of any compounddescribed herein (e.g., provide a physiologically-effective bloodprofile) over about 4, about 8, about 12, about 16 or about 24 hours.

Non-limiting examples of pharmaceutically-acceptable excipients can befound, for example, in Remington: The Science and Practice of Pharmacy,Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, JohnE., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton,Pa. 1975; Liberman, H. A. and Lachman, L., Eds., Pharmaceutical DosageForms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical DosageForms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams &Wilkins 1999), each of which is incorporated by reference in itsentirety.

Multiple therapeutic agents can be administered in any order orsimultaneously. In some embodiments, a compound of the invention isadministered in combination with, before, or after an antibiotic. Ifsimultaneously, the multiple therapeutic agents can be provided in asingle, unified form, or in multiple forms, for example, as multipleseparate pills. The agents can be packed together or separately, in asingle package or in a plurality of packages. One or all of thetherapeutic agents can be given in multiple doses. If not simultaneous,the timing between the multiple doses can vary to as much as about amonth.

Therapeutic agents described herein can be administered before, during,or after the occurrence of a disease or condition, and the timing ofadministering the composition containing a therapeutic agent can vary.For example, the compositions can be used as a prophylactic and can beadministered continuously to subjects with a propensity to conditions ordiseases in order to lessen a likelihood of the occurrence of thedisease or condition. The compositions can be administered to a subjectduring or as soon as possible after the onset of the symptoms. Theadministration of the therapeutic agents can be initiated within thefirst 48 hours of the onset of the symptoms, within the first 24 hoursof the onset of the symptoms, within the first 6 hours of the onset ofthe symptoms, or within 3 hours of the onset of the symptoms. Theinitial administration can be via any route practical, such as by anyroute described herein using any formulation described herein. Atherapeutic agent can be administered as soon as is practicable afterthe onset of a disease or condition is detected or suspected, and for alength of time necessary for the treatment of the disease, such as, forexample, from about 1 month to about 3 months. The length of treatmentcan vary for each subject.

Pharmaceutical compositions described herein can be in unit dosage formssuitable for single administration of precise dosages. In unit dosageform, the formulation is divided into unit doses containing appropriatequantities of one or more compounds. The unit dosage can be in the formof a package containing discrete quantities of the formulation.Non-limiting examples are packaged injectables, vials, or ampoules.Aqueous suspension compositions can be packaged in single-dosenon-reclosable containers. Multiple-dose reclosable containers can beused, for example, in combination with or without a preservative.Formulations for injection can be presented in unit dosage form, forexample, in ampoules, or in multi-dose containers with a preservative.

Pharmaceutical compositions provided herein, can be administered inconjunction with other therapies, for example, chemotherapy, radiation,surgery, anti-inflammatory agents, and selected vitamins. The otheragents can be administered prior to, after, or concomitantly with thepharmaceutical compositions.

Depending on the intended mode of administration, the pharmaceuticalcompositions can be in the form of solid, semi-solid or liquid dosageforms, such as, for example, tablets, suppositories, pills, capsules,powders, liquids, suspensions, lotions, creams, or gels, for example, inunit dosage form suitable for single administration of a precise dosage.

For solid compositions, nontoxic solid carriers include, for example,pharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharin, talc, cellulose, glucose, sucrose, and magnesiumcarbonate.

Non-limiting examples of pharmaceutically active agents suitable forcombination with compositions of the disclosure include anti-infectives,i.e., aminoglycosides, antiviral agents, antimicrobials,anticholinergics/anti spasmotics, antidiabetic agents, antihypertensiveagents, antineoplastics, cardiovascular agents, central nervous systemagents, coagulation modifiers, hormones, immunologic agents,immunosuppressive agents, and ophthalmic preparations.

Compounds can be delivered via liposomal technology. The use ofliposomes as drug carriers can increase the therapeutic index of thecompounds. Liposomes are composed of natural phospholipids, and cancontain mixed lipid chains with surfactant properties (e.g., eggphosphatidylethanolamine). A liposome design can employ surface ligandsfor attaching to unhealthy tissue. Non-limiting examples of liposomesinclude the multilamellar vesicle (MLV), the small unilamellar vesicle(SUV), and the large unilamellar vesicle (LUV). Liposomalphysicochemical properties can be modulated to optimize penetrationthrough biological barriers and retention at the site of administration,and to reduce a likelihood of developing premature degradation andtoxicity to non-target tissues. Optimal liposomal properties depend onthe administration route: large-sized liposomes show good retention uponlocal injection, small-sized liposomes are better suited to achievepassive targeting. PEGylation reduces the uptake of the liposomes by theliver and spleen, and increases the circulation time, resulting inincreased localization at the inflamed site due to the enhancedpermeability and retention (EPR) effect. Additionally, liposomalsurfaces can be modified to achieve selective delivery of theencapsulated drug to specific target cells. Non-limiting examples oftargeting ligands include monoclonal antibodies, vitamins, peptides, andpolysaccharides specific for receptors concentrated on the surface ofcells associated with the disease.

Non-limiting examples of dosage forms suitable for use in the disclosureinclude liquid, elixir, nanosuspension, aqueous or oily suspensions,drops, syrups, and any combination thereof. Non-limiting examples ofpharmaceutically-acceptable excipients suitable for use in thedisclosure include granulating agents, binding agents, lubricatingagents, disintegrating agents, sweetening agents, glidants,anti-adherents, anti-static agents, surfactants, anti-oxidants, gums,coating agents, coloring agents, flavoring agents, coating agents,plasticizers, preservatives, suspending agents, emulsifying agents,plant cellulosic material and spheronization agents, and any combinationthereof.

Compositions of the invention can be packaged as a kit. In someembodiments, a kit includes written instructions on theadministration/use of the composition. The written material can be, forexample, a label. The written material can suggest conditions methods ofadministration. The instructions provide the subject and the supervisingphysician with the best guidance for achieving the optimal clinicaloutcome from the administration of the therapy. The written material canbe a label. In some embodiments, the label can be approved by aregulatory agency, for example the U.S. Food and Drug Administration(FDA), the European Medicines Agency (EMA), or other regulatoryagencies.

Dosing.

Pharmaceutical compositions described herein can be in unit dosage formssuitable for single administration of precise dosages. In unit dosageform, the formulation is divided into unit doses containing appropriatequantities of one or more compounds. The unit dosage can be in the formof a package containing discrete quantities of the formulation.Non-limiting examples are liquids in vials or ampoules. Aqueoussuspension compositions can be packaged in single-dose non-reclosablecontainers. Multiple-dose reclosable containers can be used, forexample, in combination with a preservative. Formulations for parenteralinjection can be presented in unit dosage form, for example, inampoules, or in multi-dose containers with a preservative.

A compound described herein can be present in a composition in a rangeof from about 1 mg to about 2000 mg; from about 100 mg to about 2000 mg;from about 10 mg to about 2000 mg; from about 5 mg to about 1000 mg,from about 10 mg to about 500 mg, from about 50 mg to about 250 mg, fromabout 100 mg to about 200 mg, from about 1 mg to about 50 mg, from about50 mg to about 100 mg, from about 100 mg to about 150 mg, from about 150mg to about 200 mg, from about 200 mg to about 250 mg, from about 250 mgto about 300 mg, from about 300 mg to about 350 mg, from about 350 mg toabout 400 mg, from about 400 mg to about 450 mg, from about 450 mg toabout 500 mg, from about 500 mg to about 550 mg, from about 550 mg toabout 600 mg, from about 600 mg to about 650 mg, from about 650 mg toabout 700 mg, from about 700 mg to about 750 mg, from about 750 mg toabout 800 mg, from about 800 mg to about 850 mg, from about 850 mg toabout 900 mg, from about 900 mg to about 950 mg, or from about 950 mg toabout 1000 mg.

A compound described herein can be present in a composition in an amountof about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 10mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg,about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg,about 95 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg,about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg,about 450 mg, about 500 mg, about 550 mg, about 600 mg, about 650 mg,about 700 mg, about 750 mg, about 800 mg, about 850 mg, about 900 mg,about 950 mg, about 1000 mg, about 1050 mg, about 1100 mg, about 1150mg, about 1200 mg, about 1250 mg, about 1300 mg, about 1350 mg, about1400 mg, about 1450 mg, about 1500 mg, about 1550 mg, about 1600 mg,about 1650 mg, about 1700 mg, about 1750 mg, about 1800 mg, about 1850mg, about 1900 mg, about 1950 mg, or about 2000 mg.

In some embodiments, a dose can be expressed in terms of an amount ofthe drug divided by the mass of the subject, for example, milligrams ofdrug per kilograms of subject body mass. In some embodiments, a compoundis administered in an amount ranging from about 5 mg/kg to about 50mg/kg, 250 mg/kg to about 2000 mg/kg, about 10 mg/kg to about 800 mg/kg,about 50 mg/kg to about 400 mg/kg, about 100 mg/kg to about 300 mg/kg,or about 150 mg/kg to about 200 mg/kg.

EXAMPLES Example 1: Synthesis of 2,3-di-((E)-2-arylethenyl)indoles

2,3-di-((E)-2-Arylethenyl)indoles (compounds 1-6) were prepared usingoxidative Heck coupling as detailed in Scheme 1.

Palladium acetate (0.1 eq) was added to a mixture of a selected styrene(4 eq), copper (II) acetate (4 eq), and indole (1 eq) in DMF/DMSO (9:1).The reaction mixture was stirred at 70-80° C. for 18-24 hours withthin-layer chromatography (TLC) monitoring of the reaction progress (20%EtOAc/hexanes). The reaction was cooled to room temperature andpartitioned between minimal amounts of water and EtOAc, which wasfiltered through a plug of celite. The layers were then separated andthe organic layer was washed with saturated brine solution, dried overMgSO₄, filtered, and concentrated under reduced pressure. Flashchromatography afforded the desired 2,3-di-((E)-2-arylethenyl)indoles asdepicted below:

Yield 41%; ¹H NMR (400 MHz, DMSO-d₆) δ 11.60 (s, 1H), 8.09 (d, J=8.0 Hz,1H), 7.80 (ddd, J=16.5, 7.8, 4.6 Hz, 6H), 7.49 (d, J=8.3 Hz, 2H), 7.42(dd, J=8.3, 6.6 Hz, 3H), 7.31-7.19 (m, 3H), 7.13 (t, J=7.5 Hz, 1H); ¹³CNMR (100 MHz, DMSO-d₆) δ: 138.9, 138.8, 137.1, 136.7, 133.6, 132.3,129.7, 129.4, 129.0, 128.3, 127.7, 127.3, 125.9, 124.4, 123.0, 121.7,121.3, 118.2, 115.4, 112.1; HRMS calculated for C₂₄H₁₈Cl₂N (M+H)⁺:390.0816, actual HRMS 390.0787.

Yield 60%; ¹H NMR (400 MHz, DMSO-d₆) δ 11.56 (s, 1H), 8.07 (d, J=8.6 Hz,1H), 7.77 (q, J=10.6 Hz, 7H), 7.48-7.18 (m, 14H), 7.12 (s, 1H); ¹³C NMR(100 MHz, DMSO-d₆) δ: 139.0, 138.1, 137.4, 136.4, 129.2, 128.9, 128.8,128.2, 127.1, 126.9, 126.4, 126.1, 125.8, 123.6, 121.8, 121.2, 120.5,117.0, 113.9, 111.6; HRMS calculated for C₂₄H₂₀N (M+H)⁺: 322.1596,actual HRMS 322.1606.

Yield 66%; ¹H NMR (400 MHz, DMSO-d₆) δ 10.57 (s, 1H), 8.25-7.83 (m, 1H),7.83 6.69 (m, 7H), 4.20-2.48 (m, 6H); ¹³C NMR (100 MHz, DMSO-d₆) δ:158.8, 158.6, 131.6, 127.8, 127.6, 127.0, 126.6, 125.8, 124.6, 124.4,123.9, 122.9, 121.8, 120.5, 120.2, 119.8, 119.6, 119.2, 114.1, 113.9,111.6, 111.6, 54.6; HRMS calculated for C₂₆H₂₄NO₂ (M+H)⁺: 382.1807,actual HRMS 382.1800.

Yield 49%; ¹H NMR (400 MHz, DMSO-d₆) δ 10.68 (s, 1H), 8.09 (d, J=7.9 Hz,1H), 7.81-7.69 (m, 6H), 7.46-7.39 (m, 1H), 7.37-7.10 (m, 8H); ¹³C NMR(100 MHz, DMSO-d₆) δ: 137.7, 135.5, 135.4, 133.8, 128.4, 128.3, 127.5,127.5, 126.8, 125.0, 123.3, 121.2, 121.1, 120.7, 120.2, 116.4, 115.6,115.4, 115.3, 115.0, 111.1, 111.0; HRMS calculated for C₂₄H₁₈F₂N (M+H)⁺:358.1407, actual HRMS 358.1407.

Yield 40%; ¹H NMR (400 MHz, DMSO-d₆) δ 11.07 (s, 1H), 8.07 (q, J=8.9,8.4 Hz, 3H), 7.87 (td, J=15.7, 2.1 Hz, 2H), 7.72-7.58 (m, 2H), 7.51-7.18(m, 10H); ¹³C NMR (100 MHz, DMSO-d₆) δ: 137.1, 136.5, 134.7, 134.6,133.3, 133.0, 130.0, 129.8, 128.8, 127.8, 127.1, 126.9, 126.7, 126.2,125.8, 124.3, 124.2, 123.5, 122.7, 121.1, 120.8, 120.2, 118.6, 115.7,110.9; HRMS calculated for C₂₄H₁₈Cl₂N (M+H)⁺: 390.0816, actual HRMS390.0815.

Yield 40%; ¹H NMR (400 MHz, acetone-d₆) δ 10.79 (s, 1H), 8.14 (dd,J=7.9, 3.2 Hz, 1H), 7.99-7.87 (m, 2H), 7.76 (dd, J=13.1, 2.6 Hz, 2H),7.69-7.55 (m, 3H), 7.48-7.14 (m, 11H), ¹³C NMR (100 MHz, acetone-d₆) δ:135.8, 134.2, 134.1, 130.3, 130.0, 127.3, 126.7, 126.1, 125.8, 125.4,125.3, 124.9, 124.5, 123.6, 122.8, 120.9, 120.4, 118.0, 114.6, 111.2;HRMS calculated for C₂₄H₁₈Cl₂N (M+H)⁺: 390.0816, actual HRMS 390.0807.

¹H NMR (400 MHz, acetone-d₆) δ 11.09 (s, 1H), 8.66-8.64 (d, J=7.28 Hz,1H), 8.22-8.20 (d, J=8.0 Hz, 2H), 7.91-7.90 (d, J=7.28 Hz, 2H),7.50-7.48 (d, J=6.8 Hz, 1H), 7.28-7.08 (m, 5H); ¹³C NMR (100 MHz,acetone-d₆) δ: 161.5, 161.3, 149.6, 142.3, 137.0, 132.4, 131.6, 124.7,124.6, 124.1, 124.0, 123.3, 121.0, 115.1, 115.0, 112.1. HRMS calculatedfor C₂₂H₂₀N₃O₂ (M+H)⁺: 358.1556, actual HRMS 358.1556.

¹H NMR (400 MHz, acetone-d₆) δ 11.20 (s, 1H), 8.53-8.51 (d, J=6.60 Hz,1H), 8.31 (s, 1H), 7.84-7.70 (m, 4H), 7.56-7.54 (d, J=7.24 Hz, 1H),7.31-7.30 (m, 2H); ¹³C NMR (100 MHz, acetone-d₆) δ: 152.9, 137.1, 136.4,133.4, 132.1, 124.1, 123.3, 122.7, 121.9, 118.6, 112.0. HRMS calculatedfor C₁₄H₁₁BrN₃ (M+H)⁺: 300.0136, actual HRMS 300.0135.

¹H NMR (400 MHz, acetone-d₆) δ 11.17 (s, 1H), 8.54-8.52 (d, J=7.6 Hz,1H), 8.30 (s, 1H), 7.91-7.89 (d, J=8.64 Hz, 1H), 7.56-7.54 (m, 3H),7.34-7.27 (m, 2H); ¹³C NMR (100 MHz, acetone-d₆) δ: 153.5, 138.0, 137.3,134.6, 134.2, 130.0, 125.0, 123.9, 123.6, 123.5, 119.6, 112.9. HRMScalculated for C₁₄H₁₁ClN₃ (M+H)⁺: 256.0642, actual HRMS 256.0642.

¹H NMR (400 MHz, acetone-d₆) δ 10.66 (s, 1H), 7.92-7.90 (d, J=8.52 Hz,2H), m, 1H), 7.49-6.85 (m, 10H); ¹³C NMR (100 MHz, acetone-d₆) δ: 157.4,132.6, 129.7, 129.6, 129.5, 129.3, 129.2, 129.0, 127.3, 127.2, 126.5,123.6, 123.2, 121.5, 119.9, 117.5, 117.2, 117.1, 116.8, 111.9. HRMScalculated for C₂₀H₁₄Cl₂NO (M+H)⁺: 354.0452, actual HRMS 354.0459.

¹H NMR (400 MHz, acetone-d₆) δ 10.85 (s, 1H), 7.91-7.89 (d, J=7.8 Hz,2H), m, 1H), 7.50-7.05 (m, 9H); ¹³C NMR (100 MHz, acetone-d₆) δ: 158.0,134.0, 133.6, 130.8, 130.5, 130.1, 130.0, 128.3, 127.8, 127.7, 126.1,124.2, 123.4, 118.0, 117.5, 114.4. HRMS calculated for C₂₀H₁₃Cl₃NO(M+H)⁺: 388.0063, actual HRMS 388.0067.

Non-limiting examples of compounds of the invention include thefollowing:

and pharmaceutically-acceptable salts thereof.

Example 2: Toxicity of Compounds of the Invention on HeLa and WI38 Cells

The toxicities of the compounds of the invention were determined forhuman cancerous cervical epithelium HeLa and normal lung fibroblast WI38cells. The toxicities of EPIs INF-55 and INF-55Cl were quantified ascontrols.

TABLE 1 shows the measured toxicities (IC₅₀; μM) of the compounds of theinvention and the EPIs INF-55 and INF-55Cl against HeLa cells. TABLE 2compares the toxicity of compounds 7-11 on HeLa cells and normal WI38cells. The results indicated that compounds 7, 9, and 10 killedcancerous HeLa cells at concentrations that were not toxic to normalWI38 cells in culture.

TABLE 1 Compound IC₅₀ HeLa 1 68.9 ± 4.8 7 38.55 +/− 2.52 8 31.56 +/−1.74 9 34.37 +/− 0.85 10 36.82 +/− 1.53 11 25.85 +/− 2.47 13 16.4 ± 1.114 12.2 ± 1.6 15 Non-Toxic 16  6.4 ± 1.0 18 14.3 ± 0.7 19 35.8 ± 6 INF-55 Non-Toxic 20 Non-Toxic 21  8.2 ± 1.0 22  3.2 ± 0.4 23 12.9 ± 3.224 23.9 ± 1.8 25  13 ± 3.5 26 18.6 ± 1.1 27 12.5 ± 0.2 28  8.6 ± 1.4 2911.6 ± 0.7 30 34.4 ± 5.0 31 Non-Toxic INF-55Cl Non-Toxic

TABLE 2 Compound IC₅₀ “cancer” HeLa (μM) IC₅₀ “normal” WI38 (μM) 7 38.55+/− 2.52 53.03 +/− 9.84 8 31.56 +/− 1.74 11.67 +/− 0.82 9 34.37 +/− 0.8561.89 +/− 6.63 10 36.82 +/− 1.53  54.81 +/− 12.99 11 25.85 +/− 2.47 9.62 +/− 0.24

Example 3: Minimum Inhibitory Concentrations (MICs) of Compounds of theInvention and Antibiotics Against MRSA Isolates

The MICs of S. aureus ATCC 33591, BAA-44, BAA-1707, BAA-1717, BAA-1720,BAA-1747, BAA-1754, BAA-1761, BAA-1763, BAA-1764, and BAA-1766 weremeasured using a cell concentration of about 5×10⁵ colony forming units(CFU)/mL. In a 96-well microtiter plate, two-fold dilutions were made ofeach drug (starting with 100 CPM) in 100 μL of a cell suspension intrypticase soy broth (TSB). The samples were incubated overnight at 37°C. on a rotary shaking incubator set at 100 revolutions per minute (rpm)and were visually inspected for turbidity. A 20% well-volume of3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTTreagent, 5 mg/mL) was added, and the samples were incubated for about 20minutes. The MICs were determined as the lowest concentration at whichfull visual inhibition was observed by the unaided eye.

TABLE 3 shows the MICs of compound 1 and compounds 7-9 against variousMRSA isolates. TABLE 4 shows the MICs of antibiotics, includingoxacillin, norfloxacin, tetracycline, gentamycin, vancomycin,erythromycin, and penicillin, against various MRSA isolates. The resultsin TABLE 4 indicated that several of the MRSA isolates developedresistance against the antibiotics, which are indicated with *.

TABLE 3 MRSA Isolate (ATCC) 1 (μM) 7 (μM) 8 (μM) 9 (μM) 10 (μM) 11 (μM)33591 TBD  50-100 3.1-6.3 12.5 12.5  6.3-12.5 BAA-44 400 100 15 16 8 4BAA-1707 TBD  6.3-12.5 6.3 12.5 12.5 12.5 BAA-1717 TBD 12.5 6.3 6.3-12.5 6.3 3.1-6.3 BAA-1720 TBD 12.5-25   6.3 12.5 6.3 3.1-6.3BAA-1747 TBD  50-100 6.3 12.5 12.5-25    6.3-12.5 BAA-1754 TBD >100 12.512.5 50  50-100 BAA-1761 TBD 50 12.5 12.5 >100  50-100 BAA-1763 TBD >1006.3 12.5  50-100 50 BAA-1764 TBD >100 12.5 12.5 >100 50 BAA-1766TBD >100 12.5 12.5  25-50  50-100

TABLE 4 MRSA Isolate Oxacillin Norfloxacin Tetracycline GentamycinVancomycin Erythromycin Penicillin (ATCC) (μg/mL) (μg/mL) (μg/mL)(μg/mL) (μg/mL) (μg/mL) (mg/mL) 33591 >100*    25*   100* >100* <3.1 TBDTBD BAA-44   400*   100*    3.1 >200* 3.1 >1000* 1.6* BAA-1707  <3.1 <3.1    25*  <3.1 <3.1 TBD TBD BAA-1717   200* TBD  <1.6    3.1 <1.6   30*   1* BAA-1720 >100* >100*  <3.1  <3.1 <3.1 TBD TBD BAA-1747   6.3    6.3  <3.1    6.3 <3.1 TBD TBD BAA-1754    50*  <3.1  <3.1 <3.1 <3.1 TBD TBD BAA-1761    6.3 >100*    12.5  <3.1 <3.1 TBD TBDBAA-1763    50* >100*  <3.1 3-6 <3.1 TBD TBD BAA-1764    25*  <3.1  <3.1 <3.1 <3.1 TBD TBD BAA-1766   <6.3  <3.1  <3.1    12.5* <3.1 TBD TBD*indicates development of resistance

Example 4: Fluorescent Competition Assay with Compound 1 with MRSA andS. aureus

A fluorescent binding assay was performed to demonstrate that MRSA hasmore binding sites for compound 1 than S. aureus due to theoverexpression of NorA efflux pumps in MRSA (FIG. 1). The error barsrepresent data from three independent measurements.

S. aureus and MRSA cells (about 10⁸ CFU/mL) were treated with two dosesof compound 1 (1 μM and 10 μM), and the cells were incubated at 37° C.for 15 minutes on a rotary shaker set at 150 rpm. After the incubation,the cells were transferred to a 96-well fluorescent plate reader andread every minute for 30 minutes at excitation wavelengths of 334 nm,380 nm, and 407 nm, and an emission wavelength of 508 nm. The controlswere: 1×PBS, compound 1 (1 M and 10 μM) in 1×PBS, S. aureus in 1×PBS,and MRSA in 1×PBS.

The results in FIG. 1 indicated that MRSA was able to bind more ofcompound 1, as shown by the overall higher level of fluorescence whencompared to the binding of compound 1 by S. aureus.

Example 5: Fluorescent Binding Assay of Compound 1 Versus INF-55 andReserpine

INF-55 and reserpine, NorA EPIs, were used to compete with compound 1for binding sites on MRSA. Since compound 1 is highly fluorescent,binding could be assessed by measuring the fluorescence at excitationwavelengths of 345 nm, 380 nm, 407 nm, and an emission wavelength of 508nm. Fluorescent readings were taken every minute for 30 minutes at alamp energy of 20,000 CW. MRSA cells (about 10⁸ CFU/mL) were pelleted at7500 rpm and resuspended in 1×PBS. The MRSA cells were then pretreatedwith the appropriate EPI at varying concentrations and incubated for 30minutes at 37° C. at 150 rpm on a rotary shaker.

INF-55 (FIG. 2) and reserpine (FIG. 3) were added at 1 μM, 10 μM, 50 μM,and 100 μM. After incubation with the appropriate EPI, compound 1 wasadded at 5 μM and the samples were incubated for 10 minutes. The sampleswere then placed in a fluorescent plate reader for analysis. Thecontrols used were as follows: 1×PBS, compound 1 (5 μM) in 1×PBS, eachrespective concentration of the EPI in 1×PBS, compound 1 (5 μM) added toMRSA cells, and each respective EPI added to MRSA cells. The instrumentbaseline fluorescence drift was corrected by subtracting thefluorescence measured at each time point for the 1×PBS control sample.The error bars represent data from three independent measurements.

The results from FIG. 2 indicated that compound 1 was able to competewith INF-55 for binding to the efflux pumps of MRSA, as shown by thefluorescence of compound 1.

The results from FIG. 3 indicated that compound 1 was able to competewith reserpine for binding to the efflux pumps of MRSA, as shown by thefluorescence of compound 1.

Example 6: Light-Activated Killing of Bacteria Using Compounds of theInvention Using Photodynamic Therapy

Some compounds herein exhibited antibacterial activity upon exposure tolight and showed toxicity to a large number drug-resistant bacterialspecies. Photodynamic therapy (PDT) with compound 1 reduced 100,000,000viable MRSA cells to 0 viability upon receiving 2 minutes of white lightirradiation. The compounds of the invention were effective at killing awide variety of human, animal and agricultural pathogens, including CRE(gut, urinary tract and wound infections), S. pyogenes (flesh-eatingbacteria), S. mutans (dental caries), Clavibacter (a major agriculturalpathogen), and antibiotic-sensitive organisms. Gram-negative bacteria(e.g., A. baumannii) were sensitized to these PDT actions usingnon-toxic concentrations of PMB.

Bacterial media was inoculated with bacteria and incubated overnight.The bacterial cultures were diluted to 5×10⁸ CFU/mL using a McFarlandlatex turbidity standard (0.5). Compound 1 was diluted with sterileDMSO, which was added to the cell suspension in a glass culture tube.The cells were then incubated for 30 minutes at 37° C. on a rotaryshaking incubator at 150 rpm. 500 μL of the treated cell suspension wasadded to a well on a sterile ceramic drop plate. Non-coherent whitelight from a Lumacare™ LC-122 unit was used to irradiate the samples.The end of the light probe was placed 3 cm above the sample well,perpendicular to the surface of the sample, and light was applied for 2minutes (15 seconds light, 15 seconds dark). 200 μL of the irradiatedsample was transferred to a 96-well plastic microtiter plate, and fiveten-fold dilutions were made in the media. 10 μL of each dilution wasdripped onto an agar media plate and streaked down the plate by tiltingthe plate down. The agar plates were incubated at 37° C. overnight, andcolony counts were performed to calculate the CFU/mL.

FIG. 4 depicts the effects of PDT on MRSA using a hand-held flashlight,which can either be UV or plain white-light emitted using a LumaCare™instrument, a hand-held white-light emitting flashlight, or a hand-heldChauvet® LED mini strobe light. The image on the left shows thePDT-stimulated clearance of MRSA, and the image on the right shows ascanning electron microscope image of cells upon being treated with acompound of the invention and receiving PDT. The SEM image shows thatwhile cells treated with the combination of compound 9 at 25 uM andlight using a LumaCare™ instrument appear to have burst from the inside.The cells treated with either light or compound 9 alone were undamaged.

Example 7: Light-Activated Killing of Gram-Positive Organisms withCompound 1

Bacterial patterning is a visually illustrative approach used todemonstrate control of bacterial growth with light and a photo-activatedcompound. To determine the light-activated killing of Gram-positivebacteria using light and compound 1, an agar plate was preparedcontaining 20 μM of inactive and non-irradiated compound 1. 20 μM ofcompound 1 was used to prevent the inhibition of bacterial growthbecause the 20 μM concentration was significantly lower than the MICvalue (400 μM). A chessboard mask was then placed on top of the agarplate. The agar plate was then irradiated with white light tophoto-activate compound 1 only in the exposed areas (dark squares ofFIG. 5), which caused inhibition of bacterial growth in the illuminatedparts of the agar plate, where compound 1 was photoactivated. The agarplate was then inoculated with bacteria and incubated overnight at 37°C. Bacterial colonies were observed only where the area of the plate wascovered to prevent irradiation as seen in FIG. 5.

Dose-dependent effects of treating bacteria with compound 1 andirradiation with white light were assessed by treating 10⁸ CFU/mL ofcells with compound 1, followed by irradiation with white light for 2minutes. Compound 1 was added at a specific concentration to 1 mL of a10⁸ CFU/mL suspension of the respective bacterial cells, and the cellswere incubated at 37° C. for approximately 45 minutes on a rotary shakerat 150 rpm. 0.5 mL of the cell suspension was removed and placed intothe wells of a sterile ceramic drop plate, and then the cells wereirradiated with white light from a Lumacare™ LC 122A light source for 2minutes at a distance of approximately 3 cm. The time and distance ofthe light application delivered about 85 J/cm² of 400-700 nm light tothe sample. After being irradiated, ten-fold dilutions were made of eachcell sample. The dilutions were drip-streaked onto a tryptic soy agar(TSA) plate and incubated at 37° C. for 18-24 hours. Enumeration ofbacteria was determined from the dilution, which produced a drip-streakcontaining approximately 30-300 colonies. After counting the colonies,the CFU/mL was calculated by applying the respective dilution factor anda factor of 100 to account for the 10 μL of sample taken. Appropriatecontrols were prepared, including cells with no treatment, cells withonly compound 1 added, and cells only treated with light.

TABLE 5 and FIG. 6 show the light-activated killing of MRSA (ATCC No.BAA-44; hospital-acquired MRSA). The error bars in FIG. 6 represent thevariations between three replicate determinations. The results indicatedthat treatment with light led to greater cell toxicity than treatment ofthe cells with compound 1 alone.

TABLE 5 1 - Dark Toxicity 1 - Light Toxicity Treatment (CFU/mL) (CFU/mL)No 2.67 × 10⁸ ± 6.76 × 10⁷ 2.94 × 10⁸ ± 4.08 × 10⁷ treatment 0.1 μM 2.85× 10⁸ ± 8.05 × 10⁷ 2.00 × 10³ ± 4.00 × 10² 0.5 μM 2.93 × 10⁸ ± 2.14 ×10⁷ 3.33 × 10¹ ± 5.77 × 10¹ 1.0 μM 1.05 × 10⁸ ± 9.07 × 10⁶ None detected(<100 CFU/mL) 5.1 μM 4.34 × 10⁵ ± 5.04 × 10⁵ None detected (<100 CFU/mL)

FIG. 7 also shows the light-activated killing of MRSA. The results showthat there was a 3-log reduction in the concentration of CFUs when thecells were treated with 5 μM of compound 1, and there was a 1-logreduction in the concentration of CFUs when the cells were treated with1 μM of compound 1.

FIG. 8 shows the UV/Vis spectrum of compound 1. The results show thatcompound 1 had a primary peak maximum at 331 nm and a secondary peakmaximum at 388 nm. These peaks are characteristic of and dependent onthe specific structure of compound 1. Since the toxicity of compound 1could be activated by white light alone (illustrated in FIG. 4), PDTactivation was not confined to the compound's absorbance maxima.

TABLE 6 shows the synergy and photodynamic inactivation resulting fromtreatment with compounds 1, 10, and 11. The results show that compounds1, 10, and 11 were more effective at killing cells when the compoundswere used in conjunction with norfloxacin or oxacillin. When compounds1, 10, and 11 were used to treat cells in conjunction with PDT, onlycompound 1 had an increased ability to kill cells.

TABLE 6 Synergy with Synergy with Compound Norfloxacin Oxacillin PDT 15-log reduction 5-log reduction <1-log reduction 10 5-log reduction4-log reduction N/A 11 8-log reduction 5-log reduction N/A

TABLE 7 and FIG. 9 show the light-activated killing of MRSA (ATCC No.BAA-1717; community-acquired MRSA). The error bars in FIG. 9 representthe variations between three replicate determinations. The resultsindicated that treatment with light and compound 1 led to greater celltoxicity than treatment of the cells with compound 1 alone.

TABLE 7 1 - Dark Toxicity 1 - Light Toxicity Treatment (CFU/mL) (CFU/mL)No 6.30 × 10⁷ ± 9.29 × 10⁶ 2.94 × 10⁷ ± 8.89 × 10⁶ treatment 0.1 μM 5.10× 10⁷ ± 5.29 × 10⁶ 4.43 × 10³ ± 1.33 × 10³ 0.5 μM 4.03 × 10⁶ ± 1.5 ×10⁶   4.0 × 10² ± 3.61 × 10² 1.0 μM 1.06 × 10⁶ ± 8.39 × 10⁴ Nonedetected (<100 CFU/mL) 5.1 μM 7.53 × 10⁵ ± 1.12 × 10⁵ None detected(<100 CFU/mL)

TABLE 8 and FIG. 10 show the light-activated killing of S. aureus (ATCCNo. 29213). The error bars in FIG. 10 represent the variations betweenthree replicate determinations. The results indicated that treatmentwith light and compound 1 led to greater cell toxicity than treatment ofthe cells with compound 1 alone.

TABLE 8 1 - Dark Toxicity 1 - Light Toxicity Treatment (CFU/mL) (CFU/mL)No 4.27 × 10⁷ ± 1.62 × 10⁷ 9.93 × 10⁷ ± 3.83 × 10⁷ treatment 0.1 μM 1.63× 10⁸ ± 1.72 × 10⁷ 1.20 × 10⁵ ± 5.12 × 10⁴ 0.5 μM 2.67 × 10⁶ ± 2.10 ×10⁶ 1.60 × 10³ ± 1.44 × 10³ 1.0 μM 1.34 × 10⁶ ± 3.04 × 10⁵ None detected(<100 CFU/mL) 5.1 μM 7.13 × 10⁴ ± 1.80 × 10⁴ None detected (<100 CFU/mL)

TABLE 9 and FIG. 11 show the light-activated killing of VRE (ATCC No.51299). The error bars in FIG. 11 represent the variations between threereplicate determinations. The results indicated that treatment withlight led to greater cell toxicity than treatment of the cells withcompound 1 alone.

TABLE 9 1 - Dark Toxicity 1 - Light Toxicity Treatment (CFU/mL) (CFU/mL)No 8.67 × 10⁸ ± 1.40 × 10⁸ 5.78 × 10⁸ ± 4.27 × 10⁸ treatment 0.1 μM 6.50× 10⁸ ± 5.40 × 10⁸ 7.00 × 10² ± 8.19 × 10² 0.5 μM 6.50 × 10⁸ ± 1.32 ×10⁸ 2.67 × 10² ± 4.62 × 10² 1.0 μM 6.20 × 10⁸ ± 2.62 × 10⁸ None detected(<100 CFU/mL) 5.0 μM 4.55 × 10⁵ ± 8.87 × 10⁵ None detected (<100 CFU/mL)

TABLE 10, TABLE 11, and FIG. 12 show the light-activated killing of S.pyogenes (ATCC No. 8133). TABLE 10 depicts the MICs of compound 1 andlight when used individually to treat S. pyogenes. The results in TABLE11 indicated that treatment with light and compound 1 led to greatercell toxicity than treatment of the cells with compound 1 or lightalone. The error bars in FIG. 12 represent the variations between threereplicate determinations.

TABLE 10 Drug S. pyogenes MIC Compound 1 100 μM Light No effect

TABLE 11 1 - Dark Toxicity 1- Light Toxicity Treatment (CFU/mL) (CFU/mL)No 8.00 × 10⁶ ± 1.42 × 10⁶ 7.67 × 10⁶ ± 7.51 × 10⁵ Treatment 0.1 μM 7.13× 10⁶ ± 1.10 × 10⁶ None Detected (<100 CFU/mL) 0.5 μM 1.40 × 10⁶ ± 7.05× 10⁵ None Detected (<100 CFU/mL) 1.0 μM 4.13 × 10⁶ ± 9.07 × 10⁵ NoneDetected (<100 CFU/mL) 5.0 μM 5.01 × 10⁵ ± 4.07 × 10⁵ None Detected(<100 CFU/mL)

TABLE 12, TABLE 13, and FIG. 13 show the light-activated killing of S.mutans (Ward's 85W 2357). TABLE 12 depicts the MICs of compound 1 andlight when used individually to treat S. mutans. The results in TABLE 13indicated that treatment with light and compound 1 led to greater celltoxicity than treatment of the cells with compound 1 or light alone. Theerror bars in FIG. 13 represent the variations between three replicatedeterminations.

TABLE 12 Drug S. mutans MIC Compound 1 50-100 μM Light No effect

TABLE 13 1 - Dark Toxicity 1- Light Toxicity Treatment (CFU/mL) (CFU/mL)No 2.27 × 10⁸ ± 6.35 × 10⁷  2.03 × 10⁸ ± 5.77 × 10⁶ Treatment 0.1 μM 2.6× 10⁸ ± 5.57 × 10⁷ 2.35 × 10⁵ ± 3.50 × 10⁵ 0.5 μM 1.44 × 10⁸ ± 4.46 ×10⁷  2.33 × 10³ ± 1.54 × 10³ 1.0 μM 2.1 × 10⁵ ± 1.13 × 10⁵ None Detected(<100 CFU/mL) 5.0 μM 5.5 × 10⁴ ± 2.48 × 10⁴ None Detected (<100 CFU/mL)

Example 8: Light-Activated Killing of Gram-Positive Organisms withCompound 1 and Structural Analogues of Compound 1

To illustrate the light-induced effects exhibited by structuralanalogues of compound 1, the minimum bactericidal concentration (MBC)was determined for each of the synthesized analogues. The MBC wasdefined as the minimum concentration of a compound at which thebacterial cell population is reduced to sterility (10⁶ CFU/mL reductionafter treatment).

The bacterial media was inoculated with bacteria and incubatedovernight. The bacterial cultures were diluted to 5×10⁸ CFU/mL using aMcFarland latex turbidity standard (0.5). Compound 1 was diluted withsterile DMSO, which was added to the cell suspension in glass culturetubes. The cells were then incubated for 30 minutes at 37° C. on arotary shaking incubator at 150 rpm. 500 μL of the treated cellsuspension was added to a well on a sterile ceramic drop plate.Non-coherent white light from a Lumacare™ LC-122 unit was used toirradiate the samples. The end of the light probe was placed 3 cm abovethe sample well, perpendicular to the surface of the sample, and lightwas applied for 2 minutes (15 seconds light, 15 seconds dark). 200 μL ofthe irradiated sample was transferred to a 96-well plastic microtiterplate and five ten-fold dilutions were made in the media. 10 μL of eachdilution was dripped onto an agar media plate and was allowed to streakdown the plate by tilting the plate down. The agar plates were incubatedat 37° C. overnight and colony counts were performed to calculate theCFU/mL.

TABLE 14 details the MBC values of compounds 1-6 against MRSA with andwithout irradiation with white light. Irradiation involved treatmentwith a LumaCare™ light source at 3 cm from media for 1 min.

TABLE 14 Compound MBC in the dark (μM) MBC with irradiation (μM) 1 200 12 100 0.5 3 200 0.5 4 100 0.5 5 200 0.5 6 200 1

Example 9: Light-Activated Killing of Human Cell Lines Using Compoundsof the Invention

TABLE 15 depicts the IC₅₀ of compounds in the presence or absence oflight for human cell lines. The cell lines used were HeLa (humancervical adenocarcinoma), U-87 MG (human brain gliobastoma), MES-SA(human uterine sarcoma), MES-SA/Dx5 (human uterine sarcoma grown inpresence of doxorubicin), NCI-H441 (human lung papillaryadenocarcinoma), A549 (human lung carcinoma), WI-38 (human embryoniclung normal), MCF7 (human breast adenocarcinoma), SW1088 (human brainastrocytoma), B16F10 (mouse skin melanoma), NIH-3T3 (mouse fibroblast),and Jurkat (human lymphoblastoma).

“1-Sol” in TABLE 15 was generated by dissolving compound 1 and potassiumhydroxide at a 1:1 ratio in a DMSO/water solution. The cells wereirradiated with continuous white light using a LumaCare™ light sourcefor 2 minutes at a distance of 6.5 cm from the cells. The resultsindicated that all the compounds tested became more toxic uponadministration of light regardless of whether the compounds wereadministered with or without KOH, or with or without liposomeencapsulation.

TABLE 15 Compound IC₅₀ (μM) ± Compound + light Compound Cell Line SDIC₅₀ (μM) ± SD 1 HeLa  9.43 ± 0.40 <0.4 1 HeLa + 5% FBS  2.9 ± 0.2 <0.41 HeLa >0.8 0.125 ± 0.013 1 HeLa + 5% FBS >0.8 0.075 ± 0.003 1 HeLa 9.11 ± 0.94 0.29 ± 0.02 1 U-87 MG 44.56 ± 3.68 0.56 ± 0.02 1 U-87 MG(spheroids) >40 μM 0.45 1 MES-SA 42.7 ± 3.4 0.36 ± 0.05 1 MES-SA/Dx5 67.2 ± 17.9 0.72 ± 0.07 (no doxorubicin) 1 MES-SA/Dx5 30.16 ± 2.12 0.62± 0.03 (no doxorubicin) 1 MES-SA/Dx5 + 38.78 ± 2.76 0.44 ± 0.1 doxorubicin 1 NCI-H441 50.69 ± 4.37 0.76 ± 0.07 1 A549 17.35 ± 1.52 0.84± 0.32 1 WI-38 63.35 ± 5.48 0.33 ± 0.08 1 MCF7 17.82 ± 0.22 0.33 ± 0.041 SW1088 >50 0.47 ± 0.03 1 B16F10 18.66 ± 0.99 0.41 ± 0.22 1 MCF7 17.82± 0.22 0.33 ± 0.04 1 MCF7A 24.5 ± 0.2 0.40 ± 0.01 1 SW1088 >50 0.47 ±0.03 1 B16F10 18.66 ± 0.99 0.41 ± 0.22 1-Sol HeLa  55.6 ± 1.44 0.33 ±0.04 1-Sol U-87 MG 71.23 ± 4.07 0.69 ± 0.41 4 U-87 MG 42.57 ± 5.53 1.01± 0.16 4 MES-SA/Dx5  57.2 ± 10.3 1.31 ± 0.51 (no doxorubicin) 4MES-SA/Dx5 + 40.94 ± 1.36 0.67 ± 0.01 doxorubicin 1 Jurkat >10 uM 0.025uM 1 in Jurkat >10 uM 0.050 uM liposomes

Example 10: Light-Activated Killing of Parasites Using Compounds of theInvention

The results in TABLE 16 indicate that the compounds of the inventionbecame more potent upon being exposed to light when used to treatprotozoa. Trypanosomes were cultured in LIT media+10% FBS. The compoundswere added to create a dilution series and incubated with thetrypanosomes for 1 hour. The trypanosomes were then irradiated withcontinuous white light for 2 minutes at a distance of 6.5 cm using aLumaCare™ light source. 24 hours later, an MTT assay was performed,which was confirmed by visual inspection, to determine the IC₅₀ values.

TABLE 16 Compound + light Compound Protozoan parasite IC₅₀ (μM) IC₅₀(μM) 1 Trypanosoma cruzi >100 μM <0.5 μM 1-Sol Trypanosoma cruzi >100 μM<0.5 μM 4-Sol Trypanosoma cruzi >100 μM <0.5 μM

Example 11: Synergy of Compound 1 with Antibiotics Against MRSA andGram-Negative Bacteria Depicted Via an Isobologram

The synergy of two compounds with varying relative potencies can berepresented using an isobologram. An isobologram plots the normalizedeffective concentrations of each drug on each axis, where the sum of thetwo concentrations equals the line of additivity. Outside the line ofadditivity, when the sum >1, the effect of the two drugs is consideredantagonistic; inside of the line of additivity, when the sum <1, theeffect of the two drugs is considered superadditive, or when the sum≤0.5, the effect of the two drugs is considered synergistic.

The synergy of compound 1 with vancomycin, tetracycline, doxycycline,norfloxacin, dicloxicillin, oxacillin, penicillin G, and tobramycin wastested. An illustrative example based on a single data point for eachantibiotic in combination with compound 1 against MRSA (ATCC No. BAA-44)is shown in FIG. 14. A zoomed-in isobologram for the same data as shownin FIG. 14 is depicted in FIG. 15 specifically for the synergistic area.The results indicated that compound 1 was able to synergize with everytested antibiotic.

Example 12: Synergy of Compound 1 with Antibiotics Against MRSA andGram-Negative Bacteria Depicted Via a Checkerboard Assay

Another method for determining synergy is a checkerboard assay. Acheckerboard assay is performed whereby one of the test compounds isserially diluted horizontally across a plate, while the other testcompound is serially diluted vertically down the plate. Theperpendicular serial dilutions result in combinations of the compoundsthat range in concentrations from the highest to the lowest of eachcompound added. In the present study, the checkerboard assay method wasadjusted by serially diluting one of the test compounds horizontally andadding the second drug to each well at a concentration of ¼ MIC. The24-well microtiter plate was then incubated at 37° C. for 18-24 hours ona rotary shaker incubator at 150 rpm. After incubation, MTT reagent wasadded to each well at a 10% sample volume. Synergy was determined on thelowest concentration of the combined drugs, which produced no purplecoloring. Calculation of synergy was determined by the FractionalInhibitory Concentration Index (FICI): FICI=([Drug1]_(Synergy)/[Drug1]_(MIC))+([Drug 2]_(Synergy)/[Drug2]_(MIC)). FICI≤0.5 is an indication of synergy. Several commercially availableantibiotics from mechanistically varied families tested in combinationwith compound 1 were found to have FICI values ≤0.5.

TABLE 17 details the obtained FICI values for compound 1 in combinationwith varying antibiotics against MRSA, A. baumannii, and E. coli.

TABLE 17 Bacteria Compound Antibiotic FIC Index MRSA 1 Ampicillin 0.13MRSA 1 Amoxicillin 0.3 MRSA 1 Tetracycline 0.4 MRSA 1 Dicloxicillin 0.04MRSA 1 Norfloxacin 0.3 A. baumannii 1 PMB 0.1 E. coli 1 PMB 0.2

Example 13: Treatment ofA. Baumannii with Compound 1 and PMB at FixedConcentrations

FIG. 16 demonstrates that the combination of compound 1 and PMB waseffective at killing A. baumannii, as indicated by a decrease in theconcentration of CFU compared to when either agent was used alone.

Example 14: Treatment of E. coli with Compound 1, PMB, PME, and Light atFixed Concentrations

TABLE 18 details the obtained MIC values for cells treated with compound1, light, PMB, and Polymyxin E (PME) against E. coli.

TABLE 18 Drug E. Coli MIC Compound 1 >200 μg/mL    Light No effect PMB 2μg/mL PME 2 μg/mL

FIG. 17 demonstrates that the combination of compound 1 and PMB waseffective at killing E. coli, as indicated by a decrease in CFU comparedto when either agent was used alone.

Example 15: Potentiation of Activity of Compound 1 and Structurally andMechanistically Unrelated Antibiotics Against Gram-Positive MRSA with aLow Non-Toxic Dose of MTT

The potentiation of the antibiotic activity with compound 1 wasdemonstrated against MRSA (ATCC No. BAA-44) with a dose-dependent assayusing 10 μM of compound 1 as an adjuvant. The MICs for the antibioticsin the absence of compound 1 were calculated by adjusting the cells to5×10⁵ CFU/mL with McFarland Turbidity Standard (0.5) followed by theaddition of an antibiotic. The cells were then incubated at 37° C. for18 hours on a rotary shaking incubator at 100 rpm. Finally, MTT wasadded to the cells to assess viability.

To assess the potentiation of antibiotic activity, the cells wereadjusted to 5×10⁵ CFU/mL with McFarland Turbidity Standard (0.5).Compound 1 was then added at 10 μM, and the cells were incubated at 37°C. for 45 minutes. The desired antibiotic was then added at threesub-inhibitory concentrations, and the cells were incubated at 37° C.for 18 hours on a rotary shaking incubator at 100 rpm. The samples werethen diluted ten-fold, drip streaked onto a TSA plate, and incubated for18 hours at 37° C. The resulting cell colonies were counted, and theCFU/mL values were calculated.

FIG. 18 shows the MICs of tetracycline in the presence and absence ofcompound 1, which indicated that tetracycline was more efficacious incell killing in the presence of compound 1. The arrows highlight thedifferences in CFU/mL in the presence and absence of compound 1.

FIG. 19 shows the MICs of doxycycline in the presence and absence ofcompound 1, which indicated that doxycycline was more efficacious incell killing in the presence of compound 1. The arrows highlight thedifferences in CFU/mL in the presence and absence of compound 1.

FIG. 20 shows the MICs of norfloxacin in the presence and absence ofcompound 1, which indicated that norfloxacin was more efficacious incell killing in the presence of compound 1. The arrows highlight thedifferences in CFU/mL in the presence and absence of compound 1.

FIG. 21 shows the MICs of dicloxicillin in the presence and absence ofcompound 1, which indicated that dicloxicillin was more efficacious incell killing in the presence of compound 1. The arrows highlight thedifferences in CFU/mL in the presence and absence of compound 1.

FIG. 22 shows the MICs of oxacillin in the presence and absence ofcompound 1, which indicated that oxacillin was more efficacious in cellkilling in the presence of compound 1. The arrows highlight thedifferences in CFU/mL in the presence and absence of compound 1.

FIG. 23 shows the MICs of penicillin G in the presence and absence ofcompound 1, which indicated that penicillin G was more efficacious incell killing in the presence of compound 1. The arrows highlight thedifferences in CFU/mL in the presence and absence of compound 1.

FIG. 24 shows the MICs of tobramycin in the presence and absence ofcompound 1, which indicated that tobramycin was more efficacious in cellkilling in the presence of compound 1. The arrows highlight thedifferences in CFU/mL in the presence and absence of compound 1.

FIG. 25 shows the MICs of vancomycin in the presence and absence ofcompound 1, which indicated that vancomycin was more efficacious in cellkilling in the presence of compound 1. The arrows highlight thedifferences in CFU/mL in the presence and absence of compound 1.

Example 16: Cell Killing of Gram-Negative P. aeruginosa with Compoundsof the Invention in the Presence of a Non-Toxic Concentration of PME

FIG. 26 is an example of the treatment of P. aeruginosa with compound 1and PME at fixed concentrations. The results indicated that PME was moreefficacious in cell killing when co-administered with compound 1.Treatment of cells with 20 μM of compound 1 and 0.1 μg/mL of PMEresulted in about a 2-log reduction in the CFU/mL compared to cells thatwere treated with PME alone.

FIG. 27 is an example of the treatment of P. aeruginosa with compound 7and PME at fixed concentrations. The results indicated that PME was moreefficacious in cell killing when co-administered with compound 7.Treatment of cells with 20 μM of compound 7 and 0.1 μg/mL of PMEresulted in about a 3-log reduction in the CFU/mL compared to cells thatwere treated with PME alone.

FIG. 28 is an example of the treatment of P. aeruginosa with compound 8and PME at fixed concentrations. The results indicated that PME was moreefficacious in cell killing when co-administered with compound 8.Treatment of cells with 20 μM of compound 8 and 0.1 μg/mL of PMEresulted in about a 4-log reduction in the CFU/mL compared to cells thatwere treated with PME alone.

FIG. 29 is an example of the treatment of P. aeruginosa with compound 9and PME at fixed concentrations. The results indicated that PME was moreefficacious in cell killing when co-administered with compound 9.Treatment of cells with 20 μM of compound 9 and 0.1 μg/mL of PMEresulted in about a 2-log reduction in the CFU/mL compared to cells thatwere treated with PME alone.

FIG. 30 is an example of the treatment of P. aeruginosa with compound 10and PME at fixed concentrations. The results indicated that PME was moreefficacious in cell killing when co-administered with compound 10.Treatment of cells with 20 μM of compound 10 and 0.1 μg/mL of PMEresulted in about a 2-log reduction in the CFU/mL compared to cells thatwere treated with PME alone.

FIG. 31 is an example of the treatment of P. aeruginosa with compound 11and PME at fixed concentrations. The results indicated that PME was moreefficacious in cell killing when co-administered with compound 11.Treatment of cells with 20 μM of compound 11 and 0.1 μg/mL of PMEresulted in a about 2-log reduction in the CFU/mL compared to cells thatwere treated with PME alone.

TABLE 19 details the MICs of compounds 1, 7-11, and PME and summarizesthe data presented in FIGS. 26-31. Only compound 1 was able to decreasethe CFU/mL when used in conjunction with white light to treat P.aeruginosa.

TABLE 19 Compound MIC Co-treatment with PME PDT 1 >100 μM 2-logreduction ~7-log reduction 7 >100 μM 3-log reduction N/A 8 >100 μM 4-logreduction N/A 9 >100 μM 2-log reduction N/A 10  >100 μM 2-log reductionN/A 11  >100 μM 2-log reduction N/A PME    2 μg/mL N/A N/A

Example 17: Cell Killing of Gram-Negative Klebsiella pneumoniae UsingCompounds of the Invention in the Presence or Absence of PMB

Klebsiella pneumoniae (CRE, ATCC BAA-1705) was grown in TSB overnight at37° C. and diluted to about 5×10⁵ CFU/mL using a McFarland LatexTurbidity standard (0.5). One mL of the cell suspension was transferredto a glass culture test tube. PMB was added to the cell suspensionresulting in a final concentration of 200 μg/mL of PMB. The cellsuspension was incubated at 37° C. on a rotary shaking incubator at 150rpm for 4 hours. Compounds 1, 7, 8, 9, 10, and 11 were added to the cellsuspension, resulting in a concentration of 20 μM for each of thecompounds. The cell suspensions containing the compounds were incubatedat 37° C. on a rotary shaking incubator at 150 rpm for 4 hours. Eachsample was diluted tenfold to a 10⁻⁵ dilution. Ten 10 microliters ofeach dilution from each sample was dripped onto a TSA plate and allowedto streak down the plate. The agar plates were incubated at 37° C. for18-24 hours. Colony counts were performed and CFU/mL values werecalculated.

TABLE 20 below depicts the ability of the compounds of the invention tokill CRE in the presence of PMB. The results indicate that the CFU ofCRE decreased during combination treatment with compounds of theinvention and PMB.

TABLE 20 Compound No Treatment PMB @ 0.2 μg/mL Comp @ 20 mM PMB + Comp 16.6 × 10⁸ CFU/mL 1.8 × 10⁶ CFU/mL 6.4 × 10⁸ CFU/mL 8.2 × 10³ CFU/mL 76.6 × 10⁸ CFU/mL 1.8 × 10⁶ CFU/mL 6.9 × 10⁸ CFU/mL 100 CFU/mL 8 6.6 ×10⁸ CFU/mL 1.8 × 10⁶ CFU/mL 6.6 × 10⁸ CFU/mL None Detected 9 6.6 × 10⁸CFU/mL 1.8 × 10⁶ CFU/mL 6.4 × 10⁸ CFU/mL None Detected 10 6.6 × 10⁸CFU/mL 1.8 × 10⁶ CFU/mL 7.9 × 10⁸ CFU/mL None Detected 11 6.6 × 10⁸CFU/mL 1.8 × 10⁶ CFU/mL 7.8 × 10⁸ CFU/mL None Detected

Example 18: Turbidity Comparison of MRSA Isolates Treated with Compound7 and Oxacillin

FIG. 32 depicts a turbidity comparison for MRSA (ATCC BAA-44) isolatestreated with a positive control (vancomycin), compound 7, oxacillin, anda sample treated with both compound 7 and oxacillin. The results showthat the observed turbidity decreased in the following order: notreatment>>oxacillin>>compound 7>>compound 7+oxacillin>positive control.

FIG. 33 depicts the corresponding colony concentrations for the MRSAisolates shown in FIG. 32. The arrow in FIG. 33 shows that co-treatmentof MRSA with compound 7 and oxacillin was more effective at killingcells than individual treatments with compound 7 or oxacillin.

Example 19: Synergy of Compounds 1 and Compounds 7-11 with AntibioticsAgainst Gram-Positive S. aureus

The synergy of compounds 1 and 7-11 with oxacillin and norfloxacin wastested against MRSA (ATCC BAA-44). An overnight culture of MRSA wasfirst diluted to about 5×10⁵ CFU/mL in TSB. Then, 1 mL of the dilutedbroth suspension was added to a borosilicate glass culture tube. Anappropriate volume of a compound or an antibiotic drug stock was addedto the broth suspension and mixed using a vortex mixer. Then anappropriate volume of an antibiotic was added to test synergy withcompounds 7-11. The samples were subsequently mixed using a vortexmixer, and the samples were incubated for 18 hours at 37° C. on a rotaryshaking incubator. After 18 hours, the test cultures were dilutedten-fold, 10 μL of each dilution was drip-streaked onto a TSA plate, andthe samples were incubated for 18 hours at 37° C. Colony counts werethen performed and the CFU/mL values were calculated.

FIG. 34 shows that compound 1 was more effective at killing MRSA whenused in conjunction with oxacillin or norfloxacin. The arrows indicatethe effect of co-treating cells with compound 1 and an antibiotic incomparison to treatment with compound 1 alone. Cells that receivedco-treatment with compound 1 and oxacillin or norfloxacin each had abouta 5-log reduction in the number of surviving cells (CFU/mL).

FIG. 35 shows that compound 7 was more effective at killing MRSA whenused in conjunction with oxacillin or norfloxacin at two differentconcentrations. The arrows indicate the synergistic effect of usingcompound 7 and an antibiotic in comparison to treatment with compound 7alone. Cells treated with compound 7 and oxacillin or norfloxacingenerated MICs that were six-fold and four-fold greater than cellstreated with compound 8 alone, respectively.

FIG. 36 shows that compound 8 was more effective at killing MRSA whenused in conjunction with oxacillin or norfloxacin at two differentconcentrations. The arrows indicate the synergistic effect of treatingcells with compound 8 and an antibiotic in comparison to treatment withcompound 8 alone. Cells treated with compound 8 and oxacillin ornorfloxacin generated MICs that were three-fold and two-fold greaterthan cells treated with compound 8 alone, respectively.

FIG. 37 shows that compound 9 was more effective at killing MRSA whenused in conjunction with oxacillin or norfloxacin at varyingconcentrations. The arrows indicate this synergistic effect incomparison to treatment with compound 9 in the absence of oxacillin ornorfloxacin. Cells treated with compound 9 and oxacillin or norfloxacingenerated MICs that were two-fold greater than cells treated withcompound 9 alone.

FIG. 38 shows that compound 10 was more effective at killing MRSA whenused in conjunction with oxacillin or norfloxacin at varyingconcentrations. The arrow indicates this synergistic effect incomparison to cell treatment with compound 10 alone. While compound 10alone was able to effectively kill cells (MIC of 16 μM), cells treatedwith compound 10 and oxacillin or norfloxacin generated MICs that wereeight-fold greater than cells treated with compound 10 alone.

FIG. 39 shows that compound 10 was more effective at killing MRSA whenused in conjunction with oxacillin or norfloxacin. The arrows indicatethe effect of co-treating cells with compound 1 and oxacillin ornorfloxacin in comparison to treatment with compound 10 alone. Cellsthat received co-treatment with compound 10 and oxacillin or norfloxacinhad about a 4-log and about a 5-log reduction in the number of survivingcells (CFU/mL), respectively.

FIG. 40 shows that compound 11 was more effective at killing MRSA whenused in conjunction with oxacillin at various concentrations. The darkarrow denotes the synergistic effect in comparison to cell treatmentwith compound 11 at a concentration of 1 μM in the absence of oxacillin.

FIG. 41 shows that compound 11 was more effective at killing MRSA whenused in conjunction with oxacillin or norfloxacin. The arrows indicatethe effect of co-treating cells with compound 11 and oxacillin ornorfloxacin in comparison to treatment with compound 11 alone. Cellsthat received co-treatment with compound 11 and oxacillin or norfloxacinhad about a 5-log and about an 8-log reduction in the number ofsurviving cells (CFU/mL), respectively.

TABLE 21 details the MIC values identified in the MRSA assay ofcompounds 1, 7, 8, 9, 10, 11, various antibiotics, and light. Theresults showed that when compounds 1, 7, 8, 9, 10, and 11 were used inconjunction with oxacillin or norfloxacin, their ability to kill MRSAincreased by several orders of magnitude compared to treatment witheither the drug or compound alone at concentrations well below theirrespective MICs.

TABLE 21 Orders of Magnitude Drug MRSA MIC with Oxacillin withNorfloxacin Compound 1 400 μM >7 9 Compound 7 100 μM 6 4 Compound 8 15μM >3 >2 Compound 9 16 μM >2 >2 Compound 10 16 μM 8 8 Compound 11 8 μM 55 Dicloxicillin 500-100 μg/mL — — Doxycycline 3.12 μg/mL — — Norfloxacin100 μg/mL — — Oxacillin 400 μg/mL — — Tetracycline 3.12 μg/mL — —Tobramycin >5,000 μg/mL — — Vancomycin 3.12 μg/mL — — Light No effect ——

TABLE 22 details the combination effects identified in the MRSA assaysof combining compounds 1, 7, 8, 9, 10, and 11 with antibiotics,including dicloxicillin, norfloxacin, oxacillin, tetracycline,tobramycin, vancomycin, and light.

TABLE 22 (Fraction of MIC) (Fraction of MIC) % Compound + AntibioticKill Surviving MRSA (1/40) Compound 1 (1/50) Dicloxicillin 99.99 1.00 ×10⁻⁸ (1/40) Compound 1 (1/31) Dicloxicillin 99.99 1.00 × 10⁻⁴ (1/40)Compound 1 (1/5) Norfloxacin 99.99 1.00 × 10⁻⁹ (1/40) Compound 1 (1/4)Oxacillin 99.99 1.00 × 10⁻⁷ (1/40) Compound 1 (1/30) Tetracycline 99.991.00 × 10⁻⁵ (1/40) Compound 1 (1/50) Tobramycin 99.99 1.00 × 10⁻⁹ (1/40)Compound 1 (1/6) Vancomycin 99.99 1.00 × 10⁻⁹ (1/40) Compound 1 Light99.99 1.00 × 10⁻⁸ (1/7) Compound 7 (1/4) Oxacillin 99.99 1.00 × 10⁻⁶(1/4) Compound 7 (1/7) Norfloxacin 99.99 1.00 × 10⁻⁴ (1/3) Compound 8(1/2) Oxacillin 99.9 1.00 × 10⁻³ (1/3) Compound 8 (1/4) Norfloxacin 991.00 × 10⁻² (1/2) Compound 9 (1/3) Oxacillin 99 1.00 × 10⁻² (1/2)Compound 9 (1/4) Norfloxacin 99 1.00 × 10⁻² (1/8) Compound 10 (1/8)Oxacillin 99.99 1.00 × 10⁻⁸ (1/8) Compound 10 (1/16) Norfloxacin 99.991.00 × 10⁻⁸ (1/8) Compound 11 (1/4) Oxacillin 99.99 1.00 × 10⁻⁵ (1/8)Compound 11 (1/7) Norfloxacin 99.99 1.00 × 10⁻⁵

Example 20: Synergy of Compounds 7-11 with Antibiotics AgainstGram-Positive Enterococcus faecalis

TABLE 23 details the MIC values of compounds 1, 7, 8, 9, 10, 11, variousantibiotics, and light against VRE.

TABLE 23 Drug VRE MIC Compound 1 >200 μM Compound 7 >200 μM Compound 8 2μM Compound 9 >200 μM Compound 10 10-20 μM Compound 11 20 μMDicloxicillin 4 μg/mL Doxycycline 0.25-0.5 μg/mL Norfloxacin 2.5 μg/mLOxacillin 20-40 μg/mL Tetracycline 1.25 μg/mL Tobramycin >200 μg/mLVancomycin >40 μg/mL Light No effect

Possible synergy of compounds 7-11 with vancomycin, tetracycline, andnorfloxacin was tested against VRE (ATCC 51299). For these experiments,overnight cultures of VRE were diluted to about 5×10⁵ CFU/mL in TSB.Then, 1 mL of the diluted broth suspension was added to a borosilicateglass culture tube. After appropriate volumes of the compounds wereadded to the broth suspensions, the samples were mixed using a vortexmixer, and an appropriate volume of an antibiotic was added to thesamples. The samples were mixed using a vortex mixer and incubated for18 hours at 37° C. on a rotary shaking incubator. After 18 hours, thetest cultures were diluted ten-fold, and 10 μL of each dilution wasdrip-streaked onto a TSA plate and incubated for 18 hours at 37° C.Colony counts were then performed and the CFU/mL values were calculated.

FIG. 42 shows that compound 7 was more effective at killing VRE when thecells were treated with 75 μM of compound 7 in conjunction withvancomycin, tetracycline, or norfloxacin compared to treatment withcompound 7 or the antibiotics alone.

FIG. 43 shows that compound 8 was more effective at killing VRE when thecells were treated with 7.5 μM of compound 8 in conjunction withvancomycin, tetracycline, or norfloxacin compared to when the cells weretreated with compound 8 or the antibiotics alone.

FIG. 44 shows that compound 9 was more effective at killing VRE when thecells were treated with 75 μM of compound 9 in conjunction withtetracycline or norfloxacin compared to treatment with compound 9 or theantibiotics alone.

FIG. 45 shows that compound 10 was more effective at killing VRE whenthe cells were treated with 2-2.5 μM of compound 10 in conjunction withvancomycin, tetracycline, dicloxicillin, or norfloxacin compared totreatment with compound 10 or the antibiotics alone.

FIG. 46 shows that compound 11 was more effective at killing VRE whenthe cells were treated with 2-2.5 μM of compound 11 in conjunction withvancomycin, tetracycline, or norfloxacin compared to treatment withcompound 11 or the antibiotics alone.

TABLE 24 details the MICs of common antibiotics against VRE. In a 12×75mm borosilicate glass culture tube, 1 mL of cell suspension was added.Then, compound 7 was added to the cells, and the samples were mixedusing a vortex mixer. Then, vancomycin (4 μg/mL), norfloxacin (4 μg/mL),tetracycline (4 μg/mL), or gentamycin (4 μg/mL) were added, and thesamples were mixed using a vortex mixer. The samples were incubatedovernight at 37° C. on a rotary shaking incubator at 100 rpm. Inhibitionobserved visually compared to each individual treatment was consideredsynergistic.

The results in TABLE 24 show that in VRE, Compound 7 potentiates theactivity of vancomycin. In VRE, which is penicillin G-, ampicillin-,tetracycline- and norfloxacin-sensitive, compound 7 synergizes withvancomycin at a concentration of 4 ug/ml (CLSI vancomycin in vitrosusceptibility breakpoint: ≤2 μg/mL). S stands for ‘sensitive’ and Rstands for ‘resistant’.

TABLE 24 Antibiotic MIC S/R Penicillin G 2 μg/mL S Ampicillin 2-4 μg/mLS Vancomycin 64 μg/mL R Erythromycin >32 μg/mL R Tetracycline <0.5 μg/mLS Norfloxacin 1 μg/mL S

Example 21: Synergy of Compounds 7-11 with Antibiotics AgainstGram-Negative Klebsiella pneumoniae Using PMB

The MICs of PMB and compounds 7-11 were determined against Klebsiellapneumoniae (CRE, ATCC BAA-1705). Based on the MIC determinations, asub-inhibitory concentration of PMB (200 ng/mL) was added to the cellsuspensions, and the samples were incubated for 3 hours. After theincubation with PMB, each of the compounds was added at a concentrationof 20 μM, and the samples were incubated for an additional 18 hours. Asample was taken and diluted ten-fold in media. Ten microliters of eachdilution was then drip-streaked onto an agar plate and incubatedovernight at 37° C. Colony counts were then performed and theconcentrations of cells (CFU/mL) were calculated.

FIG. 47 shows the synergy of compounds 7-11 with PMB against CRE. Whenused with PMB, compound 7 substantially reduced the number of CREcolonies. Simultaneous treatment of cells with PMB and compounds 8-11resulted in significant antibacterial activity and reduced the bacterialpopulation to sterility.

Example 22: Synergy of Compound 7 with Antibiotics Against Gram-NegativeE. cloacae and K. pneumoniae Using PMB and PME

TABLE 25 details the MICs of PMB and PME against E. cloacae and K.pneumoniae. The results show that E. cloacae and K. pneumoniae hadidentical MICs against PME. In contrast, the MIC of PMB against K.pneumoniae was higher than that of E. cloacae and had MICs of ≥16 μg/mLand 4 μg/mL, respectively.

TABLE 25 E. cloacae K. pneumoniae Antibiotic ATCC BAA-2341 ATCC BAA-2341PME 16 μg/mL    16 μg/mL PMB  4 μg/mL ≥16 μg/mL

TABLE 26 details the combination effects of treating E. coli, E.cloacae, and K. pneumoniae with compound 7 and PMB or PME and theconditions under which the combination effects were determined. Theresults show that E. coli and K. pneumoniae exhibited additionalinhibition when treated with PMB or PME. E. cloacae exhibited additionalinhibition when treated with PME.

TABLE 26 CRE Isolate Synergy Conditions E. coli Yes [PMB] = 0.05 μg/mL(Pre-inc. for 2 hrs) + Compound 7 ATCC BAA-2340 [PME] = 0.05 μg/mL(Pre-inc. for 1 hr) + Compound 7 E. cloacae Yes [PME] = 0.50 μg/mL(Pre-inc. for 1 hr) + ATCC BAA-2341 Compound 7 K. pneumoniae Yes [PMB] =0.75 μg/mL (Pre-inc. for 2 hrs) + Compound 7 ATCC BAA-2342 [PME] = 0.75μg/mL (Pre-inc. for 2 hrs) + Compound 7

Example 23: Synergy of Compounds 8-11 with Antibiotics AgainstGram-Negative Klebsiella pneumoniae Using PME

TABLE 27 details the individual MIC values of compounds 1, 7, 8, 9, 10,11, PMB, PME, and light against CRE.

TABLE 27 Drug CRE MIC Compound 1 >100 μM Compound 7 >100 μM Compound8 >100 μM Compound 9 >100 μM Compound 10 >100 μM Compound 11 >100 μM PMB   4 μg/mL PME    4 μg/mL Light No effect

The MICs of PME and compounds 8-11 were determined against Klebsiellapneumoniae (CRE, ATCC BAA-1705). Based on the MIC determinations, asub-inhibitory concentration of PME (200 ng/mL) was added to the cellsuspensions, and the samples were incubated for 1 hour. After theincubation with PME, the compounds were added at a concentration of 20μM, and the samples were further incubated. At specific time points, thesamples were taken and diluted ten-fold in media. Ten microliters ofeach dilution was then drip-streaked onto an agar plate and incubatedovernight at 37° C. Colony counts were then performed and the CFU/mLwere calculated.

FIG. 48 shows the time-dependent changes in the colony counts of CREwhen the cells were treated with PME, compounds 8 or 9, and PME, or whenthe cells received no treatment (NT). Both compounds 8 and 9 reduced theCRE colony populations to sterility when the cells were simultaneouslytreated with PME (0.2 μg/mL). These results demonstrate the synergisticeffects of the compounds and PME.

FIG. 49 shows the time-dependent changes in the colony counts of CREupon treatment with PME, compounds 10 or 11, and PME, or when the cellsreceived no treatment (NT). Both compounds reduced the CRE colonypopulations to sterility when the cells were simultaneously treated withPME (0.2 μg/mL). These results demonstrate the synergistic effects ofthe compounds and PME.

Example 24: Synergy of Compounds 8-11 with Antibiotics Against A.baumannii (ATCC 15151) Using PME

TABLE 28 details the individual MIC values of compounds 1, 7, 8, 9, 10,11, PMB, PME, and light against A. baumannii.

TABLE 28 Drug A. baumannii MIC Compound 1 >100 μM Compound 7 >100 μMCompound 8 >100 μM Compound 9 >100 μM Compound 10 >100 μM Compound11 >100 μM PMB    4 μg/mL PME   0.5 μg/mL Light No effect

The MICs of PME and compounds 8-11 were determined against A. baumannii(ATCC 15151). Based on the MIC determinations, a sub-inhibitoryconcentration of PME (200 ng/mL) was added to the cell suspensions, andthe resulting samples were incubated for 1 hour. After incubating thecells with PME, a compound (5 μM) was added, and the samples werefurther incubated at 37° C. At specific time points, samples were takenand diluted ten-fold in media. Ten microliters of each dilution was thendrip-streaked onto an agar plate and incubated overnight at 37° C.Colony counts were then performed, and the CFU/mL were calculated.

FIG. 50 shows the MICs of compounds 7 and 9 for several time points.When the bacterial colonies were treated with compounds 7 or 9 alongwith 0.2 μg/mL of PME, the A. baumannii colony populations were reducedto sterility. The arrows indicate that the bacterial colonies did notrecover with time upon being treated with compounds 7 or 9 and PME.

FIG. 51 shows the MIC of compound 8 for several time points. When thebacterial colonies were treated with compound 8 along with PME or PMB,the A. baumannii colony populations were reduced to sterility. Thearrows indicate that the bacterial colonies did not recover with timeupon being treated with compound 8 and PME or PMB.

FIG. 52 shows the MICs of compounds 10 and 11 for several time points.When the bacterial colonies were treated with compounds 10 or 11 alongwith 0.2 μg/mL of PME, the A. baumannii colony populations were reducedto sterility. The arrows indicate that the bacterial colonies did notrecover with time upon being treated with compounds 10 or 11 and PME.

Example 25: Resensitization of Pathogenic Bacteria to Antibiotics in thePresence of Compound 1 and Compounds 7-11

The data in TABLE 29 demonstrate that the compounds of the inventionresensitized pathogenic drug-resistant bacteria to antibiotics. Thecolumn on the right shows the order-of-magnitude increase in killingefficacy when the compounds were co-administered with an antibiotic, ascompared to the more active of either of the components alone.

The combination effects of compound 1 and compounds 7-11 were quantifiedin the presence of antibiotics, including PMB, PME, oxacillin,norfloxacin, vancomycin, tetracycline, dicloxacillin, tobramycin,doxycycline, and/or light. In a 96-well microtiter plate, two-folddilutions were made of each drug (starting with 100 μM) in 100 μL of acell suspension in TSB. The resulting samples were incubated overnightat 37° C. on a rotary shaking incubator at 100 rpm. The samples werethen visually inspected for turbidity. A 20% well volume of MTT reagent(5 mg/mL) was subsequently added, and the samples were incubated forabout 20 minutes. The MICs of the compounds of the invention weredetermined as the concentrations at which full visual inhibition wasobserved.

TABLE 29 Antibiotic/Second Drug Log Compound Organism SubstanceReduction  1 (1 μM) E. Coli PMB (0.1 μg/mL) 4  1 (1 μM) E. Coli PMB (0.1μg/mL) + Light (2 min) 7  1 (1 μM) MRSA ATCC BAA-44 Light (2 min) 8  1(1 μM) MRSA ATCC BAA- Light (2 min) 6 1717  1 (1 μM) S. aureus Light (2min) 6 ATCC 29213  1 (1 μM) VRE Light (2 min) 8-9  1 (1 μM) S. pyogenesLight (2 min) 6-7 ATCC 8133  1 (1 μM) S. mutans Light (2 min) 5-6 Ward's85W 2357  1 (5 μM) A. baumannii PMB (0.5 μg/mL) 8  1 (5 μM) A. baumanniiPME (0.2 μg/mL) 8  1 (5 μM) CRE PMB (0.2 μg/mL) + Light (2 min) 3  1 (5μM) S. aureus Tetracycline (0.1 μg/mL) 5 ATCC 29213  1 (5 μM) S. aureusNorfloxacin (0.5 μg/mL) 6 ATCC 29213  1 (10 μM) MRSA Vancomycin (0.5μg/mL) 9  1 (10 μM) MRSA Tetracycline (0.1 μg/mL) 5-6  1 (10 μM) MRSADoxycycline (0.1 μg/mL) 4  1 (10 μM) MRSA Norfloxacin (20 μg/mL) 9  1(10 μM) MRSA Oxacillin (100 μg/mL) 7-8  1 (10 μM) MRSA Dicloxacillin (1μg/mL) 8  1 (10 μM) MRSA Tobramycin (100 μg/mL) 9  1 (10 μM) CRE PME(0.1 μg/mL) 9  1 (10 μM) CRE PME (0.2 μg/mL) + Light (2 min) 9  1 (20μM) P. aeruginosa PME (0.1 μg/mL) 2  1 (20 μM) P. aeruginosa PME (0.1μg/mL) + Light (2 min) 8  7 (5 μM) A. baumannii PME (0.2 μg/mL) 8  7 (15μM) MRSA Oxacillin (100 μg/mL) 6  7 (15 μM) MRSA Norfloxacin (15 μg/mL)4  7 (15 μM) MRSA Oxacillin (100 μg/mL) 6  7 (15 μM) MRSA Norfloxacin(15 μg/mL) 4  7 (20 μM) P. aeruginosa PME (0.1 μg/mL) 3  7 (20 μM) CREPMB (0.2 μg/mL) 4  7 (75 μM) VRE Norfloxacin (2.5 μg/mL) 4-5  7 (75 μM)VRE Vancomycin (3.5 μg/mL) 3-4  7 (75 μM) VRE Tetracycline (1.5 μg/mL) 3 8 (5 μM) MRSA Oxacillin (50 μg/mL) 3-4  8 (5 μM) MRSA Norfloxacin (25μg/mL) 2-3  8 (5 μM) A. baumannii PME (0.05 μg/mL) 9  8 (7.5 μM) VRENorfloxacin (2.5 μg/mL) 4-5  8 (7.5 μM) VRE Vancomycin (3.5 μg/mL) 3  8(7.5 μM) VRE Tetracycline (1.5 μg/mL) 3-4  8 (20 μM) CRE PMB (0.2 μg/mL)6  8 (20 μM) CRE PME (0.2 μg/mL) 6  8 (20 μM) A. baumannii PMB (0.2μg/mL) 9  8 (20 μM) P. aeruginosa PME (0.1 μg/mL) 4  9 (5 μM) A.baumannii PME (0.2 μg/mL) 8  9 (8 μM) MRSA Oxacillin (125 μg/mL) 2-3  9(8 μM) MRSA Norfloxacin (25 μg/mL) 2-3  9 (20 μM) CRE PMB (0.2 μg/mL) 6 9 (20 μM) CRE PME (0.2 μg/mL) 6  9 (20 μM) P. aeruginosa PME (0.1μg/mL) 2  9 (75 μM) VRE Norfloxacin (2.5 μg/mL) 4  9 (75 μM) VRETetracycline (1.5 μg/mL) 3-4 10 (1 μM) MRSA Oxacillin (100 μg/mL) 4-5 10(1 μM) MRSA Norfloxacin (15 μg/mL) 5-6 10 (2 μM) VRE Norfloxacin (5μg/mL) 5-6 10 (2 μM) VRE Tetracycline (1 μg/mL) 3-4 10 (2 μM) VREDicloxacillin (10 μg/mL) 5 10 (2.5 μM) VRE Vancomycin (3.5 μg/mL) 4 10(5 μM) A. baumannii PME (0.2 μg/mL) 8 10 (20 μM) CRE PME (0.2 μg/mL) 610 (20 μM) CRE PME (0.2 μg/mL) 6 10 (20 μM) P. aeruginosa PME (0.1μg/mL) 2 11 (0.5 μM) MRSA Oxacillin (100 μg/mL) 5 11 (0.5 μM) MRSANorfloxacin (20 μg/mL) 8 11 (0.5 μM) VRE Norfloxacin (2.5 μg/mL) 3-4 11(0.5 μM) VRE Vancomycin (3.5 μg/mL) 3 11 (0.5 μM) VRE Tetracycline (1.5μg/mL) 3 11 (5 μM) A. baumannii PME (0.2 μg/mL) 8 11 (20 μM) CRE PME(0.2 μg/mL) 6 11 (20 μM) CRE PME (0.2 μg/mL) 6 11 (20 μM) P. aeruginosaPME (0.1 μg/mL) 2

Example 26: Synergistic Effects of Treating MRSA Isolates with Compounds7-11 and Antibiotics

TABLES 30-40 show the combination effects of treating various strains ofS. aureus (i.e., ATCC 33591, BAA-44; BAA-1707; BAA-1717; BAA-1720;BAA-1747; BAA-1754; BAA-1761; BAA-1763; BAA-1764; BAA-1766) withcompounds 7-11 and an antibiotic (i.e., oxacillin, norfloxacin,tetracycline, gentamycin, and vancomycin). The results in TABLES 30-40show that compounds 7-11 were more effective at killing MRSA isolateswhen used in conjunction with an antibiotic.

Inhibition resulting from co-treatment of a broth suspension with acompound of the invention and an antibiotic was compared to brothsuspensions subjected to each individual treatment. A visual observationof inhibition with the unaided eye upon co-treatment of the brothsuspensions, based on turbidity, was considered synergistic. The MICswere read as the lowest concentration of an antimicrobial agent thatcompletely inhibited growth of the organism in the tubes ormicrodilution wells as detected by the unaided eye.

Exceptions to reading complete inhibition of growth includedGram-positive cocci. For Gram-positive cocci, trailing growth wasobserved. For these species, the MICs were read at the first spot wheretrailing was observed, and tiny buttons of growth were ignored.

Exceptions to reading complete inhibition of growth also includedtrimethoprim and sulfonamides. Antagonists in the medium allowed forsome slight growth; thus, the end point was read as the concentration inwhich there was ≥80% reduction in growth compared to the control. When asingle skipped well (i.e., wells that exhibit no growth although growthoccurs at higher concentrations) was observed, the highest MIC was read.

A compound of the invention and an antibiotic were considered to have“no synergy” when no change was observed in turbidity betweenco-treatment of a broth suspension with a compound of the invention andan antibiotic and either of the individual components alone.

MIC interpretive standards were used to classify antibiotics, includingoxacillin, norfloxacin, gentamycin, vancomycin, and tetracycline, as“susceptible”, “intermediate”, or “resistant”. Two-fold dilutions of theantibiotics were prepared in a liquid growth medium dispensed in testtubes. For broth dilutions, cation-adjusted Mueller-Hinton broth wasused to determine the MICs; cation-adjusted Mueller-Hinton brothsupplemented with 2% NaCl was used to determine the MIC of oxacillin.For agar dilutions, Mueller-Hinton agar was used to determine the MICs;Mueller-Hinton agar supplemented with 2% NaCl was used to determine theMIC of oxacillin. The antibiotic-containing tubes were inoculated with astandard bacterial suspension of 5×10⁵ CFU/mL. Following an overnightincubation (16-20 hrs) at 35±2° C. under ambient air, the tubes wereexamined for visible bacterial growth based on turbidity. The lowestconcentration of antibiotic that prevented growth was determined to bethe MIC. An incubation time of 24 hrs was used to determine the MICs ofoxacillin and vancomycin.

TABLE 30 MRSA ATCC BAA-1707 Compound Oxacillin Norfloxacin TetracyclineGentamycin Vancomycin 7 Susceptible Susceptible Synergy SusceptibleSusceptible 8 Susceptible Susceptible No Synergy Susceptible Susceptible9 Susceptible Susceptible No Synergy Susceptible Susceptible 10Susceptible Susceptible Synergy Susceptible Susceptible 11 SusceptibleSusceptible Synergy Susceptible Susceptible

TABLE 31 MRSA ATCC BAA-1717 Results Compound Oxacillin NorfloxacinTetracycline Gentamycin Vancomycin 7 Synergy Susceptible SusceptibleSusceptible Susceptible 8 Synergy Susceptible Susceptible SusceptibleSusceptible 9 Synergy Susceptible Susceptible Susceptible Susceptible 10Synergy Susceptible Susceptible Susceptible Susceptible 11 SynergySusceptible Susceptible Susceptible Susceptible

TABLE 32 MRSA ATCC BAA-1720 Results Compound Oxacillin NorfloxacinTetracycline Gentamycin Vancomycin 7 Synergy No Synergy SusceptibleSusceptible Susceptible 8 Synergy No Synergy Susceptible SusceptibleSusceptible 9 Synergy No Synergy Susceptible Susceptible Susceptible 10No Synergy No Synergy Susceptible Susceptible Susceptible 11 No SynergyNo Synergy Susceptible Susceptible Susceptible

TABLE 33 MRSA ATCC BAA-1747 Results Compound Oxacillin NorfloxacinTetracycline Gentamycin Vancomycin 7 Susceptible Susceptible SusceptibleSusceptible Susceptible 8 Susceptible Susceptible SusceptibleSusceptible Susceptible 9 Susceptible Susceptible SusceptibleSusceptible Susceptible 10 Susceptible Susceptible SusceptibleSusceptible Susceptible 11 Susceptible Susceptible SusceptibleSusceptible Susceptible

TABLE 34 MRSA ATCC BAA-44 Results Compound Oxacillin NorfloxacinTetracycline Vancomycin 7 Synergy No Synergy Susceptible Susceptible 8No Synergy No Synergy Susceptible Susceptible 9 No Synergy No SynergySusceptible Susceptible 10 No Synergy No Synergy Susceptible Susceptible11 Synergy No Synergy Susceptible Susceptible

TABLE 35 MRSA ATCC 33591 Results Compound Oxacillin NorfloxacinTetracycline Gentamycin Vancomycin 7 Synergy Synergy Susceptible SynergySusceptible 8 No Synergy No Synergy No Synergy No Synergy Susceptible 9No Synergy No Synergy No Synergy No Synergy Susceptible 10 No Synergy NoSynergy No Synergy No Synergy Susceptible 11 No Synergy No Synergy NoSynergy No Synergy Susceptible

TABLE 36 MRSA ATCC BAA-1754 Results Compound Oxacillin NorfloxacinTetracycline Gentamycin Vancomycin 7 Synergy Susceptible SusceptibleSusceptible Susceptible 8 Synergy Susceptible Susceptible SusceptibleSusceptible 9 No Synergy Susceptible Susceptible Susceptible Susceptible10 No Synergy Susceptible Susceptible Susceptible Susceptible 11 NoSynergy Susceptible Susceptible Susceptible Susceptible

TABLE 37 MRSA ATCC BAA-1761 Results Compound Oxacillin NorfloxacinTetracycline Gentamycin Vancomycin 7 Susceptible No Synergy SusceptibleSusceptible Susceptible 8 Susceptible No Synergy Susceptible SusceptibleSusceptible 9 Susceptible No Synergy Susceptible Susceptible Susceptible10 Susceptible No Synergy Susceptible Susceptible Susceptible 11Susceptible No Synergy Susceptible Susceptible Susceptible

TABLE 38 MRSA ATCC BAA-1763 Results Compound Oxacillin NorfloxacinTetracycline Gentamycin Vancomycin 7 Synergy No Synergy SusceptibleSusceptible Susceptible 8 Synergy No Synergy Susceptible SusceptibleSusceptible 9 Synergy No Synergy Susceptible Susceptible Susceptible 10No Synergy No Synergy Susceptible Susceptible Susceptible 11 No SynergyNo Synergy Susceptible Susceptible Susceptible

TABLE 39 MRSA ATCC BAA-1764 Results Compound Oxacillin NorfloxacinTetracycline Gentamycin Vancomycin 7 Synergy Susceptible SusceptibleSusceptible Susceptible 8 Synergy Susceptible Susceptible SusceptibleSusceptible 9 Synergy Susceptible Susceptible Susceptible Susceptible 10No Synergy Susceptible Susceptible Susceptible Susceptible 11 No SynergySusceptible Susceptible Susceptible Susceptible

TABLE 40 MRSA ATCC BAA-1766 Results Compound Oxacillin NorfloxacinTetracycline Gentamycin Vancomycin 7 Synergy Susceptible SusceptibleSusceptible Susceptible 8 Synergy Susceptible Susceptible SusceptibleSusceptible 9 Synergy Susceptible Susceptible Susceptible Susceptible 10No Synergy Susceptible Susceptible Susceptible Susceptible 11 No SynergySusceptible Susceptible Susceptible Susceptible

TABLE 41 summarizes the data presented in TABLES 5-17 with respect tocompound 7. These data demonstrate that compound 7 reverts a broadspectrum of MRSA strains to antibiotic-sensitive S. aureus.

The drug concentrations used to obtain these data were 2.3 μg/mL ofcompound 7 (1/6 IC₅₀ on HeLa cells); 2 μg/mL oxacillin, or 4 μg/mL ofnorfloxacin, tetracycline, and gentamycin.

TABLE 41 MRSA Isolate (ATCC) Oxacillin Norfloxacin TetracyclineGentamycin 33591 Synergy Synergy — Synergy BAA-44 Synergy No Synergy — —BAA-1707 — — Synergy — BAA-1717 Synergy — — — BAA-1720 Synergy NoSynergy — — BAA-1747 — — — — BAA-1754 Synergy — — — BAA-1761 — NoSynergy — — BAA-1763 Synergy No Synergy — — BAA-1764 Synergy — — —BAA-1766 Synergy — — —

Example 27: Light-Activated Killing of Gram-Negative Organisms withCompound 1 in the Presence of a Non-Toxic Concentration of PMB

FIG. 53 is an example of the treatment of A. baumannii with compound 1and PMB at fixed concentrations with and without irradiation using whitelight (λ). The results indicated that PMB was more efficacious in cellkilling when co-administered with compound 1 and light.

FIG. 54 is an example of the treatment of E. coli with compound 1 andPMB at fixed concentrations with and without irradiation using whitelight (λ). The results indicated that PMB was more efficacious in cellkilling when co-administered with compound 1 and light.

Example 28: Light-Activated Killing of Gram-Negative P. aeruginosa withCompounds of the Invention in the Presence of a Non-Toxic Concentrationof PME

P. aeruginosa cells were also treated with white light upon beingincubated with compound 1 for different durations of time. In a 12×75 mmborosilicate glass culture tube, 1 mL of cell suspension was added. PMEwas added to the cells, and the samples were mixed using a vortex mixer.The resulting samples were incubated for 1.5 hours at 37° C. on a rotaryshaking incubator at 100 rpm. Compound 1 was then added, and the sampleswere incubated for 30 minutes at 37° C. on a rotary shaking incubator at100 rpm. The samples were then irradiated with white light using aLumacare™ LC-122 for 2 minutes (irradiation λ₁) and incubated overnightat 37° C. The samples were irradiated again with white light using aLumacare™ LC-122 for 2 minutes (irradiation λ₂). Ten-fold dilutions weremade of each sample, and 10 μL of each dilution was drip-streaked ontoan agar plate. The samples were incubated overnight at 37° C., andcolony counts were performed to calculate CFU/mL.

FIG. 55 is an illustrative example of the treatment of P. aeruginosawith compound 1, PME, co-treatment with compound 1 and PME, andco-treatment with compound 1, PME, and light (λ₁ and λ₂) at fixedconcentrations. The results indicated that treatment with lightimmediately after a 30 minute incubation with compound 1 (λ₁) resultedin about an 8-log reduction in the CFU/mL compared to cells thatreceived co-treatment with compound 1 and PME. Cells that receivedtreatment with additional irradiation after an overnight incubation withcompound 1 (λ₂) resulted in a negligible reduction in the CFU/mLcompared to cells that received co-treatment with compound 1 and PME.

Example 29: Comparison of Compound 1 Administered to HeLa Cells in 0.1%Volume DMSO with Compound 1 Encapsulated in Liposomes

To determine if liposomes had an effect on the activity of compound 1,HeLa cells were preincubated either with compound 1 in DMSO or indipalmitoylphosphatidylcholine (DPPC) liposomes at a 1 μM finalconcentration of compound 1. An effective period of pre-incubation withthe drug in the dark was determined to be about 28 hours for compound 1in DMSO and 60 hours for liposomal delivery of compound 1. The cellswere then irradiated with continuous white light for 2 minutes at adistance of 6.5 cm using a LumaCare™ light source. An MTT assay wascarried out to evaluate cell viability 48 hours later.

The results in TABLE 42 indicate that compound 1 was able to kill HeLacells when in a DMSO solution or encapsulated in liposomes.Additionally, compound 1 in DMSO was more stable at 40° C. compared tothe liposomal formulation.

The stability of the formulation was determined using dynamic lightscattering (DLS) to measure the mean size and standard deviation, bothof which remained consistent. The experiments were repeated after theindicated periods, and the outcome of compound 1 photodynamic therapywas essentially the same in terms of cell killing.

TABLE 42 Compound 1 pre- incubation Vehicle/ Compound Compound 1 + tomax Stability at concentration 1 light killing 40° C. Compound 1 6%killing 77% killing 28 hours >2 months (1 μM) in DMSO Compound 1 9%killing 56% killing 60 hours >2 weeks (1 μM) in liposomes

Example 30: Production of Singlet Oxygen Using a Compound of theInvention

FIG. 56 depicts the ability of a compound of the invention to be aphotodynamic agent via the production of singlet oxygen species invitro. The production of singlet oxygen upon irradiation with light (hv)was detected using Singlet Oxygen Sensor Green (SOSG). 20 μM of each1-Sol and SOSG were prepared in water and allowed to equilibrate at roomtemperature for one hour. The samples were read with a UV/Vis platespectrophotometer using excitation and emission wavelengths of 500 nmand 540 nm, respectively, before and after three minutes of irradiationwith non-coherent light (400-700 nm) for three minutes at a 3 cmdistance using a Lumacare™ LC-122. The results indicated that theaddition of compound 1-Sol increased the production of singlet oxygen asmeasured by the increase in relative fluorescence units (RFU) of SOSG.

Example 31: Suppression of the Evolution of Resistance in Drug SensitiveS. aureus (ATCC 29213)

The MICs of antibiotics and compounds 7-11 were determined against S.aureus (ATCC 29213). In a 96-well microtiter plate, 100 μL of a cellsuspension (about 5×10⁵ CFU/mL in TSB media) were diluted two-fold intriplicate with compounds 7-11 or an antibiotic. Each mixture wasincubated at 37° C. on a rotary shaking incubator for about 16 hours.MTT was then added to assess the viability of the cells (at 10% wellvolume). To evaluate the cells for their evolution of resistance,sub-inhibitory concentrations (0.5 MIC; 0.25 MIC) of each compound andantibiotic were incubated with cells overnight (18-24 hrs) at 37° C. ona rotary shaking incubator at 100 rpm. The cells were then streaked ontoa TSA plate and incubated overnight at 37° C. These steps were repeatedto reach a total of 30 exposures.

TABLE 43 details the individual MIC values of compounds 1, 7, 8, 9, 10,11, various antibiotics, and light against S. aureus.

TABLE 43 Drug S. aureus MIC Compound 1 100 μM Compound 7 32 μM Compound8 8 μM Compound 9 16 μM Compound 10 32-64 μM Compound 11 32-64 μMDicloxicillin 50-100 μg/mL Doxycycline 3.12 μg/mL Norfloxacin 1.25-2.5μg/mL Oxacillin 0.5 μg/mL Tetracycline 2.5 μg/mL Tobramycin 0.25 μg/mLVancomycin 2 μg/mL Light No effect

The ability of S. aureus to develop resistance against compounds 7-11was tested using oxacillin and norfloxacin as controls. TABLE 22 showsthe MIC values for compounds 7-11 and antibiotics (i.e., oxacillin andnorfloxacin) after serial passaging at sub-inhibitory concentrations.The results show that no evolution of resistance was observed even aftera month of continuous sub-lethal exposure to compounds 7-11. Sensitivityto most compounds remained the same or decreased.

FIG. 57 and FIG. 58 are the corresponding images to TABLE 44.

TABLE 44 Initial MIC (μM) Final MIC (μM) Compound Mar. 10, 2015 Apr. 16,2015 Resistance Evolved 7 32 8 No 8 8 8 No 9 16 8 No 10 32-64 16-32 No11 32-64 >256 Yes Oxacillin 0.25 >8 Yes Norfloxacin 2 >16 Yes

Example 32: Summary of the Synergistic Activities of Compounds of theInvention

TABLE 45 summarizes the synergistic activities of the seven classes ofclinically used antibiotics and compounds of the invention against avariety of different drug-resistant bacteria.

TABLE 45 Antibiotic Families β-lactams Fluoroquinolones GlycopeptidesTetracyclines Aminoglycosides Macrolides Polypeptides Bacterial IsolateOxacillin Norfloxacin Vancoymcin Tetracycline Gentamycin ErythromycinPolymyxin E Polymyxin B MRSA ATCC Synergy (7) Synergy (7) — — Synergy(7) — — — 33591 MRSA ATCC Synergy* Synergy Synergy (7) Synergy †(1)Synergy ‡ (7) Synergy (1) — — BAA-44 (7, 8, 9, 10, 11) (7, 8, 9, 10, 11)MRSA ATCC — — — Synergy (7) — — — — BAA-1707 MRSA ATCC Synergy (7) — — —— — — — BAA-1717 MRSA ATCC Synergy (7) — — — — — — — BAA-1720 MRSA ATCCSynergy (7) — — — — — — — BAA-1747 MRSA ATCC Synergy (7) — — — — — — —BAA-1754 MRSA ATCC — — — — — — — — BAA-1761 MRSA ATCC Synergy (7) — — —— — — — BAA-1763 MRSA ATCC Synergy (7) — — — — — — — BAA-1764 MRSA ATCCSynergy (7) — — — — — — — BAA-1766 VRE ATCC Synergy Synergy SynergySynergy — — — — 51299 (10: (7, 8, 9, 10, 11) (7, 8, 9, 10, (7, 8, 9, 10,dicloxicillin) 11) 11) P. aeruginosa — — — — — — Synergy Synergy ATCC27853 (7, 8, 9, 10, (7, 8, 9, 10, 11) 11) CRE ATCC 1705 — — — — — —Synergy Synergy (7, 8, 9, 10, (7, 8, 9, 10, 11) 11) CRE ATCC 2340 — — —— — — Synergy Synergy (7, 8, 9, 10, (7, 8, 9, 10, 11) 11) CRE ATCC 2341— — — — — — Synergy Synergy (7, 8, 9, 10, (7, 8, 9, 10, 11) 11) CRE ATCC2342 — — — — — — Synergy Synergy (7, 8, 9, 10, (7, 8, 9, 10, 11) 11) A.baumannii — — — — — — Synergy Synergy ATCC 15151 (7, 8, 9, 10, (7, 8, 9,10, 11) 11) *: includes ampicillin, dicloxicillin, and penicillin G; †:includes doxycycline; ‡: includes tobramycin; —: indicates eitherexisting synergy or to be determined

Embodiments

The following non-limiting embodiments provide illustrative examples ofthe invention, but do not limit the scope of the invention.

Embodiment 1

A method of treating a condition, the method comprising administering toa subject in need thereof a therapeutically-effective amount of acompound that binds a biological structure, thereby decreasing drugresistance in a cell, wherein the compound is moretherapeutically-effective in the presence of light than in the dark.

Embodiment 2

The method of embodiment 1, wherein the subject is human.

Embodiment 3

The method of any one of embodiments 1-2, further comprising irradiatingthe compound after administration to the subject.

Embodiment 4

The method of embodiment 3, wherein the compound is irradiated withlight having a wavelength of about 200 to about 800 nm.

Embodiment 5

The method of any one of embodiments 1-4, wherein the condition iscaused by a microbe.

Embodiment 6

The method of embodiment 5, wherein the microbe is a bacterium.

Embodiment 7

The method of embodiment 5, wherein the microbe is a Gram-positivebacterium.

Embodiment 8

The method of embodiment 5, wherein the microbe is a Gram-negativebacterium.

Embodiment 9

The method of embodiment 5, wherein the microbe is a drug-resistantbacterium.

Embodiment 10

The method of embodiment 5, wherein the microbe is methicillin-resistantStaphylococcus aureus.

Embodiment 11

The method of embodiment 5, wherein the microbe is Acinetobacterbaumannii.

Embodiment 12

The method of embodiment 5, wherein the microbe is Escherichia coli.

Embodiment 13

The method of any one of embodiments 1-12, wherein the biologicalstructure is an efflux pump.

Embodiment 14

The method of any one of embodiments 1-13, wherein the compound lessensan activity of a drug resistance mechanism in the microbe.

Embodiment 15

The method of any one of embodiments 1-14, wherein the method furthercomprises administering to the subject a therapeutically-effectiveamount of a second compound.

Embodiment 16

The method of embodiment 15, wherein the compound increases an activityof the second compound.

Embodiment 17

The method of any one of embodiments 15-16, wherein the second compoundis an antibiotic.

Embodiment 18

The method of embodiment 17, wherein the antibiotic is polymyxin B or apharmaceutically-acceptable salt thereof.

Embodiment 19

The method of any one of embodiments 15-18, wherein the compound and thesecond compound are administered in a common unit dosage form.

Embodiment 20

The method of any one of embodiments 1-19, wherein the administration isoral.

Embodiment 21

The method of any one of embodiments 1-19, wherein the administration isintravenous.

Embodiment 22

The method of any one of embodiments 1-19, wherein the administration issubcutaneous.

Embodiment 23

The method of any one of embodiments 1-19, wherein the administration istopical.

Embodiment 24

The method of any one of embodiments 1-23, wherein the administrationoccurs via an oily carrier.

Embodiment 25

The method of any one of embodiments 1-24, wherein thetherapeutically-effective amount is from about 5 mg/kg to about 50mg/kg.

Embodiment 26

The method of any one of embodiments 1-25, wherein the compound is acompound of formula:

wherein:

-   -   RING is a ring system;    -   Cy¹ is a cyclic group;    -   Cy² is a cyclic group;    -   L¹ is a linking group; and    -   L² is independently a linking group, or a        pharmaceutically-acceptable salt thereof.

Embodiment 27

The method of any one of embodiments 1-26, wherein the compound is acompound of formula:

wherein:

-   -   X is N, NH, S, or O;    -   each        is independently a single bond or a double bond;    -   R¹ is H or -L¹-Cy¹;    -   R² is H or -L²-Cy²;    -   R³ is H or -L³-Cy³ and R⁴ is H or -L⁴-Cy⁴, or R³ and R⁴ together        with the atoms to which R³ and R⁴ are bound form a ring;    -   each of L¹, L², L³, and L⁴ is independently a linking group; and    -   each of Cy¹, Cy², Cy³, and Cy⁴ is independently a cyclic group,        or a pharmaceutically-acceptable salt thereof.

Embodiment 28

The method of embodiment 27, wherein the compound is a compound offormula:

Embodiment 29

The method of any one of embodiments 1-28, wherein the compound is acompound of formula:

wherein:

-   -   X is NH, S, or O;    -   Q¹ is a ring system;    -   R¹ is H or -L¹-Cy¹;    -   R² is H or -L²-Cy²;    -   each of L¹ and L² is independently a linking group; and    -   each of Cy¹ and Cy² is independently a cyclic group, or a        pharmaceutically-acceptable salt thereof.

Embodiment 30

The method of any one of embodiments 1-29, wherein the compound is acompound of formula:

wherein:

-   -   X is NH, S, or O;    -   each        is independently a single bond or a double bond;    -   R¹ is H or -L¹-Cy¹;    -   R² is H or -L²-Cy²;    -   A¹ is C(R^(1a)), C(R^(1a))(R^(1b)), N, or N(R^(1a));    -   A² is C(R^(2a)), C(R^(2a))(R^(2b)), N, or N(R^(2a));    -   A³ is C(R^(3a)), C(R^(3a))(R^(3b)), N, or N(R^(3a));    -   A⁴ is C(R^(4a)), C(R^(4a))(R^(4b)), N, or N(R^(4a));    -   each R^(1a), R^(1b), R^(2a), R^(2b), R^(3a), R^(3b), R^(4a), and        R^(4b) is independently: halogen, hydroxyl, sulfhydryl, nitro,        nitroso, cyano, azido, a sulfoxide group, a sulfone group, a        sulfonamide group, a sulfonic acid group, an imine group, an        acyl group, an acyloxy group, alkyl, alkenyl, alkynyl, an alkoxy        group, an ether group, a carboxylic acid group, a carboxaldehyde        group, an ester group, an amine group, an amide group, a        carbonate group, a carbamate group, a thioether group, a        thioester group, a thioacid group, aryl, aryloxy, arylalkyl,        arylalkoxy, heterocyclyl, heterocyclylalkyl, heteroaryl, or        heteroarylalkyl, any of which is substituted or unsubstituted,        or H, or R^(1a) and R^(1b) together form a carbonyl, a        thiocarbonyl, an imine, or an olefin, or R^(2a) and R^(2b)        together form a carbonyl, a thiocarbonyl, an imine, or an        olefin, or R^(3a) and R^(3b) together form a carbonyl, a        thiocarbonyl, an imine, or an olefin, or R^(4a) and R^(4b)        together form a carbonyl, a thiocarbonyl, an imine, or an        olefin;    -   each of L¹ and L² is independently a linking group; and    -   each of Cy¹ and Cy² is independently a cyclic group, or a        pharmaceutically-acceptable salt thereof.

Embodiment 31

The method of embodiment 30, wherein the compound is a compound offormula:

wherein

-   -   each        is independently a single, double, or triple bond; and    -   each Y¹, Y², Z¹, and Z² is independently: a bond, an alkylene        group, an alkenylene group, an alkynylene group, an amino        linkage, and ether linkage, a thioether linkage, an ester        linkage, a thioester linkage, an amide linkage, a carbamate        linkage, a carbonate linkage, a ureido linkage, a sulfoxide        linkage, a sulfone linkage, a sulfonamide linkage, or an imine        linkage.

Embodiment 32

The method of embodiment 31, wherein the compound is a compound offormula:

wherein: each R^(1a), R^(1b), R^(2a), R^(2b), R^(3a), R^(3b), R^(4a),and R^(4b) is independently: halogen, hydroxyl, sulfhydryl, nitro,nitroso, cyano, azido, a sulfoxide group, a sulfone group, a sulfonamidegroup, a sulfonic acid group, an imine group, an acyl group, an acyloxygroup, alkyl, alkenyl, alkynyl, an alkoxy group, an ether group, acarboxylic acid group, a carboxaldehyde group, an ester group, an aminegroup, an amide group, a carbonate group, a carbamate group, a thioethergroup, a thioester group, a thioacid group, aryl, aryloxy, arylalkyl,arylalkoxy, heterocyclyl, heterocyclylalkyl, heteroaryl, orheteroarylalkyl, any of which is substituted or unsubstituted, or H.

Embodiment 33

The method of embodiment 32, wherein: X is NH, Cy¹ is aryl that isunsubstituted or substituted, and Cy² is aryl that is unsubstituted orsubstituted.

Embodiment 34

The method of embodiment 32, wherein: X is NH, Cy¹ is phenyl that isunsubstituted or substituted, and Cy² is phenyl that is unsubstituted orsubstituted.

Embodiment 35

The method of embodiment 32, wherein each R^(1a), R^(2a), R^(3a), andR^(4a) is independently: F, Cl, Br, I, hydroxyl, sulfhydryl, nitro,nitroso, an acyl group, an acyloxy group, alkyl, alkenyl, alkynyl, analkoxy group, an ether group, a carboxylic acid group, an ester group,an amine group, an amide group, a carbonate group, or a carbamate group,any of which is substituted or unsubstituted, or H.

Embodiment 36

The method of embodiment 32, wherein each R^(1a), R^(2a), R^(3a), andR^(4a) is H.

Embodiment 37

The method of any one of embodiments 1-36, wherein the compound is:

or a pharmaceutically-acceptable salt thereof.

Embodiment 38

The method of any one of embodiments 1-36, wherein the compound is:

or a pharmaceutically-acceptable salt thereof.

Embodiment 39

The method of any one of embodiments 1-36, wherein the compound is:

or a pharmaceutically-acceptable salt thereof.

Embodiment 40

The method of any one of embodiments 1-36, wherein the compound is:

or a pharmaceutically-acceptable salt thereof.

Embodiment 41

A pharmaceutical composition comprising, in a unit dosage form:

a) a therapeutically-effective amount of a compound of formula:

wherein:

-   -   X is NH, S, or O;    -   each        is independently a single bond or a double bond;    -   R¹ is -L¹-Cy¹;    -   R² is -L²-Cy²;    -   A¹ is C(R^(1a)), C(R^(1a))(R^(1b)), N, or N(R^(1a));    -   A² is C(R^(2a)), C(R^(2a))(R^(2b)), N, or N(R^(2a));    -   A³ is C(R^(3a)), C(R^(3a))(R^(3b)), N, or N(R^(3a));    -   A⁴ is C(R^(4a)), C(R^(4a))(R^(4b)), N, or N(R^(4a));    -   each R^(1a), R^(1b), R^(2a), R^(2b), R^(3a), R^(3b), R^(4a), and        R^(4b) is independently: halogen, hydroxyl, sulfhydryl, nitro,        nitroso, cyano, azido, a sulfoxide group, a sulfone group, a        sulfonamide group, a sulfonic acid group, an imine group, an        acyl group, an acyloxy group, alkyl, alkenyl, alkynyl, an alkoxy        group, an ether group, a carboxylic acid group, a carboxaldehyde        group, an ester group, an amine group, an amide group, a        carbonate group, a carbamate group, a thioether group, a        thioester group, a thioacid group, aryl, aryloxy, arylalkyl,        arylalkoxy, heterocyclyl, heterocyclylalkyl, heteroaryl, or        heteroarylalkyl, any of which is substituted or unsubstituted,        or H, or R^(1a) and R^(1b) together form a carbonyl, a        thiocarbonyl, an imine, or an olefin, or R^(2a) and R^(2b)        together form a carbonyl, a thiocarbonyl, an imine, or an        olefin, or R^(3a) and R^(3b) together form a carbonyl, a        thiocarbonyl, an imine, or an olefin, or R^(4a) and R^(4b)        together form a carbonyl, a thiocarbonyl, an imine, or an        olefin;    -   each of L¹ and L² is independently a linking group; and    -   each of Cy¹ and Cy² is independently a cyclic group, or a        pharmaceutically-acceptable salt thereof; and

b) a pharmaceutically-acceptable excipient.

Embodiment 42

The pharmaceutical composition of embodiment 41, wherein the compound isa compound of formula:

-   -   each        is independently a single, double, or triple bond; and    -   each Y¹, Y², Z¹, and Z² is independently: a bond, an alkylene        group, an alkenylene group, an alkynylene group, an amino        linkage, and ether linkage, a thioether linkage, an ester        linkage, a thioester linkage, an amide linkage, a carbamate        linkage, a carbonate linkage, a ureido linkage, a sulfoxide        linkage, a sulfone linkage, a sulfonamide linkage, or an imine        linkage.

Embodiment 43

The pharmaceutical composition of any one of embodiments 41-42, whereinthe compound is a compound of formula:

wherein: each R^(1a), R^(1b), R^(2a), R^(2b), R^(3a), R^(3b), R^(4a),and R^(4b) is independently: halogen, hydroxyl, sulfhydryl, nitro,nitroso, cyano, azido, a sulfoxide group, a sulfone group, a sulfonamidegroup, a sulfonic acid group, an imine group, an acyl group, an acyloxygroup, alkyl, alkenyl, alkynyl, an alkoxy group, an ether group, acarboxylic acid group, a carboxaldehyde group, an ester group, an aminegroup, an amide group, a carbonate group, a carbamate group, a thioethergroup, a thioester group, a thioacid group, aryl, aryloxy, arylalkyl,arylalkoxy, heterocyclyl, heterocyclylalkyl, heteroaryl, orheteroarylalkyl, any of which is substituted or unsubstituted, or H.

Embodiment 44

The pharmaceutical composition of embodiment 43, wherein: X is NH, Cy¹is aryl that is unsubstituted or substituted, and Cy² is aryl that isunsubstituted or substituted.

Embodiment 45

The pharmaceutical composition of embodiment 43, wherein: X is NH, Cy¹is phenyl that is unsubstituted or substituted, and Cy² is phenyl thatis unsubstituted or substituted.

Embodiment 46

The pharmaceutical composition of embodiment 43, wherein each R^(1a),R^(2a), R^(3a), and R^(4a) is independently: F, Cl, Br, I, hydroxyl,sulfhydryl, nitro, nitroso, an acyl group, an acyloxy group, alkyl,alkenyl, alkynyl, an alkoxy group, an ether group, a carboxylic acidgroup, an ester group, an amine group, an amide group, a carbonategroup, or a carbamate group, any of which is substituted orunsubstituted, or H.

Embodiment 47

The pharmaceutical composition of embodiment 43, wherein each R^(1a),R^(2a), R^(3a), and R^(4a) is H.

Embodiment 48

The pharmaceutical composition of any one of embodiments 41-47, whereinthe compound is:

or a pharmaceutically-acceptable salt thereof.

Embodiment 49

The pharmaceutical composition of any one of embodiments 41-47, whereinthe compound is:

or a pharmaceutically-acceptable salt thereof.

Embodiment 50

The pharmaceutical composition of any one of embodiments 41-47, whereinthe compound is:

or a pharmaceutically-acceptable salt thereof.

Embodiment 51

The pharmaceutical composition of any one of embodiments 41-47, whereinthe compound is:

or a pharmaceutically-acceptable salt thereof.

Embodiment 52

The pharmaceutical composition of any one of embodiments 41-51, furthercomprising an antibiotic.

Embodiment 53

The pharmaceutical composition of embodiment 52, wherein the antibioticis polymyxin B or a pharmaceutically-acceptable salt thereof.

Embodiment 54

The pharmaceutical composition of any one of embodiments 41-53, whereinthe therapeutically-effective amount is from about 5 mg/kg to about 50mg/kg.

Embodiment 55

The pharmaceutical composition of any one of embodiments 41-54, whereinthe pharmaceutically-acceptable excipient is an oily carrier.

Embodiment 56

A method of treating a condition, the method comprising administering toa subject in need thereof a therapeutically-effective amount of acompound of formula:

wherein:

-   -   X is NH, S, or O;    -   each        is independently a single bond or a double bond;    -   R¹ is -L¹-Cy¹;    -   R² is -L²-Cy²;    -   A¹ is C(R^(1a)), C(R^(1a))(R^(1b)), N, or N(R^(1a));    -   A² is C(R^(2a)), C(R^(2a))(R^(2b)), N, or N(R^(2a));    -   A³ is C(R^(3a)), C(R^(3a))(R^(3b)), N, or N(R^(3a));    -   A⁴ is C(R^(4a)), C(R^(4a))(R^(4b)), N, or N(R^(4a));    -   each R^(1a), R^(1b), R^(2a), R^(2b), R^(3a), R^(3b), R^(4a), and        R^(4b) is independently: halogen, hydroxyl, sulfhydryl, nitro,        nitroso, cyano, azido, a sulfoxide group, a sulfone group, a        sulfonamide group, a sulfonic acid group, an imine group, an        acyl group, an acyloxy group, alkyl, alkenyl, alkynyl, an alkoxy        group, an ether group, a carboxylic acid group, a carboxaldehyde        group, an ester group, an amine group, an amide group, a        carbonate group, a carbamate group, a thioether group, a        thioester group, a thioacid group, aryl, aryloxy, arylalkyl,        arylalkoxy, heterocyclyl, heterocyclylalkyl, heteroaryl, or        heteroarylalkyl, any of which is substituted or unsubstituted,        or H, or R^(1a) and R^(1b) together form a carbonyl, a        thiocarbonyl, an imine, or an olefin, or R^(2a) and R^(2b)        together form a carbonyl, a thiocarbonyl, an imine, or an        olefin, or R^(3a) and R^(3b) together form a carbonyl, a        thiocarbonyl, an imine, or an olefin, or R^(4a) and R^(4b)        together form a carbonyl, a thiocarbonyl, an imine, or an        olefin;    -   each of L¹ and L² is independently a linking group; and    -   each of Cy¹ and Cy² is independently a cyclic group, or a        pharmaceutically-acceptable salt thereof.

Embodiment 57

The method of embodiment 56, wherein the compound is a compound offormula:

wherein:

-   -   each        is independently a single, double, or triple bond; and    -   each Y¹, Y², Z¹, and Z² is independently: a bond, an alkylene        group, an alkenylene group, an alkynylene group, an amino        linkage, and ether linkage, a thioether linkage, an ester        linkage, a thioester linkage, an amide linkage, a carbamate        linkage, a carbonate linkage, a ureido linkage, a sulfoxide        linkage, a sulfone linkage, a sulfonamide linkage, or an imine        linkage.

Embodiment 58

The method of any one of embodiments 56-57, wherein the compound is acompound of formula:

wherein: each R^(1a), R^(1b), R^(2a), R^(2b), R^(3a), R^(3b), R^(4a),and R^(4b) is independently: halogen, hydroxyl, sulfhydryl, nitro,nitroso, cyano, azido, a sulfoxide group, a sulfone group, a sulfonamidegroup, a sulfonic acid group, an imine group, an acyl group, an acyloxygroup, alkyl, alkenyl, alkynyl, an alkoxy group, an ether group, acarboxylic acid group, a carboxaldehyde group, an ester group, an aminegroup, an amide group, a carbonate group, a carbamate group, a thioethergroup, a thioester group, a thioacid group, aryl, aryloxy, arylalkyl,arylalkoxy, heterocyclyl, heterocyclylalkyl, heteroaryl, orheteroarylalkyl, any of which is substituted or unsubstituted, or H.

Embodiment 59

The method of embodiment 58, wherein: X is NH, Cy¹ is aryl that isunsubstituted or substituted, and Cy² is aryl that is unsubstituted orsubstituted.

Embodiment 60

The method of embodiment 58, wherein: X is NH, Cy¹ is phenyl that isunsubstituted or substituted, and Cy² is phenyl that is unsubstituted orsubstituted.

Embodiment 61

The method of embodiment 58, wherein each R^(1a), R^(2a), R^(3a), andR^(4a) is independently: F, Cl, Br, I, hydroxyl, sulfhydryl, nitro,nitroso, an acyl group, an acyloxy group, alkyl, alkenyl, alkynyl, analkoxy group, an ether group, a carboxylic acid group, an ester group,an amine group, an amide group, a carbonate group, or a carbamate group,any of which is substituted or unsubstituted, or H.

Embodiment 62

The method of embodiment 58, wherein each R^(1a), R^(2a), R^(3a), andR^(4a) is H.

Embodiment 63

The method of any one of embodiments 56-62, wherein the compound is:

Embodiment 64

The method of any one of embodiments 56-62 wherein the compound is:

Embodiment 65

The method of any one of embodiments 56-62, wherein the compound is:

Embodiment 66

The method of any one of embodiments 56-62, wherein the compound is:

Embodiment 67

The method of any one of embodiments 56-66, wherein thetherapeutically-effective amount is from about 5 mg/kg to about 50mg/kg.

Embodiment 68

The method of any one of embodiments 56-67, wherein the subject ishuman.

Embodiment 69

The method of any one of embodiments 56-68, further comprisingirradiating the compound after administration to the subject.

Embodiment 70

The method of embodiment 69, wherein the compound is irradiated withlight having a wavelength of about 200 to about 800 nm.

Embodiment 71

The method of any one of embodiments 56-70, wherein the administrationis oral.

Embodiment 72

The method of any one of embodiments 56-70, wherein the administrationis intravenous.

Embodiment 73

The method of any one of embodiments 56-70, wherein the administrationis subcutaneous.

Embodiment 74

The method of any one of embodiments 56-70, wherein the administrationis topical.

Embodiment 75

The method of any one of embodiments 56-74, wherein the administrationoccurs via an oily carrier.

Embodiment 76

The method of any one of embodiments 56-75, wherein the condition is acancer.

Embodiment 77

A method of reducing drug resistance in a cell, the method comprisingcontacting the cell with a therapeutically-effective amount of acompound that binds a biological structure that reduces a drugresistance mechanism in the cell, wherein the compound is moretherapeutically-effective in the presence of light than in the dark.

Embodiment 78

A method of increasing the activity of a therapeutic first compound in acell, the method comprising contacting the cell with atherapeutically-effective amount of the therapeutic first compound and atherapeutically-effective amount of a second compound, wherein thetherapeutic first compound has a therapeutic effect that is greater thanthe therapeutic effect in absence of the second compound, wherein thesecond compound is more effective in the presence of light than in thedark.

Embodiment 80

A method of treating a condition, the method comprising administering toa subject in need thereof a therapeutically-effective amount of acompound that binds a biological structure, thereby decreasing drugresistance in a cell, and a therapeutically-effective amount of a secondagent.

Embodiment 81

The method of embodiment 80, wherein the subject is human.

Embodiment 82

The method of any one of embodiments 80-81, wherein the compound thatbinds the biological structure is more effective in the presence oflight than in the dark.

Embodiment 83

The method of embodiment 82, further comprising irradiating the compoundwith light having a wavelength of about 200 to about 800 nm.

Embodiment 84

The method of any one of embodiments 80-84, wherein the condition iscaused by a microbe.

Embodiment 85

The method of embodiment 84, wherein the microbe is a bacterium.

Embodiment 86

The method of embodiment 84, wherein the microbe is Gram-positivebacterium.

Embodiment 87

The method of embodiment 84, wherein the microbe is Gram-negativebacterium.

Embodiment 88

The method of embodiment 84, wherein the microbe is drug-resistantbacterium.

Embodiment 89

The method of embodiment 84, wherein the microbe ismethicillin-resistant Staphylococcus aureus.

Embodiment 90

The method of embodiment 84, wherein the microbe is Acinetobacterbaumannii.

Embodiment 91

The method of embodiment 84, wherein the microbe is Escherichia coli.

Embodiment 92

The method of any one of embodiments 80-91, wherein the biologicalstructure is an efflux pump.

Embodiment 93

The method of any one of embodiments 80-92, wherein the compound lessensan activity of a drug resistance mechanism in the microbe.

Embodiment 94

The method of any one of embodiments 80-93, wherein the second agent isan antibiotic.

Embodiment 95

The method of any one of embodiment 94, wherein the antibiotic ispolymyxin B or a pharmaceutically-acceptable salt thereof.

Embodiment 96

The method of any one of embodiments 94-95, wherein the compound and thesecond agent are administered in a common unit dosage form.

Embodiment 97

The method of any one of embodiments 80-96, wherein the administrationis oral.

Embodiment 98

The method of any one of embodiments 80-96, wherein the administrationis intravenous.

Embodiment 99

The method of any one of embodiments 80-96, wherein the administrationis subcutaneous.

Embodiment 100

The method of any one of embodiments 80-96, wherein the administrationis topical.

Embodiment 101

The method of any one of embodiments 80-100, wherein the administrationoccurs via an oily carrier.

Embodiment 102

The method of any one of embodiments 80-101, wherein thetherapeutically-effective amount of the compound is from about 5 mg/kgto about 50 mg/kg.

Embodiment 103

The method of any one of embodiments 80-95, wherein the method furthercomprises irradiating the compound that binds a biological structure andthe antibiotic.

Embodiment 104

The method of any one of embodiment 80-95, wherein the compound is acompound of formula:

wherein:

-   -   R¹ is hydrogen or an ester group;    -   R² is hydrogen, halogen, or L¹-Ar¹;    -   R³ is hydrogen, halogen, or L²-Ar²;    -   or R² and R³ together with the atoms to which R² and R³ are        bound form a substituted or unsubstituted ring;    -   each L¹ and L² is independently a linking group or a bond;    -   Ar¹ is a substituted or unsubstituted aryl group;    -   Ar² is a substituted or unsubstituted aryl group wherein Ar² is        not substituted with an amide, amine, nitro, imine, or ester        group;    -   each A¹, A², A³, and A⁴ is independently C(R^(1a)),        C(R^(1a))(R^(1b)), N, or N(R^(1a));    -   each R^(1a) and R^(1b) is independently hydrogen, halogen,        hydroxyl, sulfhydryl, nitro, nitroso, cyano, azido, a sulfoxide        group, a sulfone group, a sulfonamide group, a sulfonic acid        group, an imine group, an acyl group, an acyloxy group, alkyl,        alkenyl, alkynyl, an alkoxy group, an ether group, a carboxylic        acid group, a carboxaldehyde group, an ester group, an amine        group, an amide group, a carbonate group, a carbamate group, a        thioether group, a thioester group, a thioacid group, aryl,        aryloxy, arylalkyl, arylalkoxy, heterocyclyl, heterocyclylalkyl,        heteroaryl, or heteroarylalkyl, any of which is substituted or        unsubstituted; and    -   each        is independently a single or double bond,        or a pharmaceutically-acceptable salt thereof, wherein the        compound is not:

Embodiment 105

The method of embodiment 104, wherein when Ar¹ is phenyl brominated atone position, then Ar² is substituted on at least one position.

Embodiment 106

The method of embodiment 104, wherein when Ar¹ is phenyl substitutedwith one methoxy group, then Ar² is substituted on at least oneposition.

Embodiment 107

The method of embodiment 104, wherein Ar¹ is substituted, and Ar² issubstituted.

Embodiment 108

The method of embodiment 104, wherein Ar¹ is unsubstituted, and Ar² isunsubstituted.

Embodiment 109

The method of embodiment 104, wherein both L¹ and L² are independently

Embodiment 110

The method of embodiment 109, wherein both Ar¹ and Ar² are independentlysubstituted with hydrogen, halogen, or alkyloxy.

Embodiment 111

The method of embodiment 104, wherein each linking group isindependently alkylene, alkenylene, O, S, SO₂, CO, N₂, or a bond.

Embodiment 112

The method of embodiment 104, wherein each

is independently chosen to provide an aromatic system.

Embodiment 113

The method of any one of embodiments 80-112, wherein the compound is:

Embodiment 114

The method of any one of embodiments 80-112, wherein the compound is:

Embodiment 115

The method of any one of embodiments 80-112, wherein the compound is:

Embodiment 116

The method of any one of embodiments 80-112, wherein the compound is:

Embodiment 117

The method of any one of embodiments 80-112, wherein the compound is:

Embodiment 120

A compound of the formula:

wherein:

-   -   R¹ is hydrogen or an ester group;    -   R² is hydrogen, halogen, or L¹-Ar¹;    -   R³ is hydrogen, halogen, or L²-Ar²;    -   or R² and R³ together with the atoms to which R² and R³ are        bound form a substituted or unsubstituted ring;    -   each L¹ and L² is independently a linking group or a bond;    -   Ar¹ is a substituted or unsubstituted aryl group;    -   Ar² is a substituted or unsubstituted aryl group wherein Ar² is        not substituted with an amide, amine, nitro, imine, or ester        group;    -   each A¹, A², A³, and A⁴ is independently C(R^(1a)),        C(R^(1a))(R^(1b)), N, or N(R^(1a));    -   each R^(1a) and R^(1b) is independently hydrogen, halogen,        hydroxyl, sulfhydryl, nitro, nitroso, cyano, azido, a sulfoxide        group, a sulfone group, a sulfonamide group, a sulfonic acid        group, an imine group, an acyl group, an acyloxy group, alkyl,        alkenyl, alkynyl, an alkoxy group, an ether group, a carboxylic        acid group, a carboxaldehyde group, an ester group, an amine        group, an amide group, a carbonate group, a carbamate group, a        thioether group, a thioester group, a thioacid group, aryl,        aryloxy, arylalkyl, arylalkoxy, heterocyclyl, heterocyclylalkyl,        heteroaryl, or heteroarylalkyl, any of which is substituted or        unsubstituted; and    -   each        is independently a single or double bond,        or a pharmaceutically-acceptable salt thereof, wherein the        compound is not:

Embodiment 121

The compound of embodiment 120, wherein when Ar¹ is phenyl brominated atone position, then Ar² is substituted on at least one position.

Embodiment 122

The compound of embodiment 120, wherein when Ar¹ is phenyl substitutedwith one methoxy group, then Ar² is substituted on at least oneposition.

Embodiment 123

The compound of embodiment 120, wherein Ar¹ is substituted, and Ar² issubstituted.

Embodiment 124

The compound of embodiment 120, wherein Ar¹ is unsubstituted, and Ar² isunsubstituted.

Embodiment 125

The compound of embodiment 120, wherein both L¹ and L² are independently

Embodiment 126

The compound of embodiment 125, wherein both Ar¹ and Ar² areindependently substituted with hydrogen, halogen, or alkyloxy.

Embodiment 127

The compound of embodiment 120, wherein each linking group isindependently alkylene, alkenylene, O, S, SO₂, CO, N₂, or a bond.

Embodiment 128

The compound of embodiment 120, wherein each

is independently chosen to provide an aromatic system.

Embodiment 129

The compound of embodiment 120-128, wherein the compound is:

Embodiment 130

The compound of embodiment 120-128, wherein the compound is:

Embodiment 131

The compound of embodiment 120-128, wherein the compound is:

Embodiment 132

The compound of embodiment 120-128, wherein the compound is:

Embodiment 133

The compound of embodiment 120-128, wherein the compound is:

Embodiment 140

The method of any one of embodiments 1-25, wherein the compound is acompound of the formula:

wherein:

-   -   R¹ is hydrogen or an ester group;    -   R² is hydrogen, halogen, or L¹-Ar¹;    -   R³ is hydrogen, halogen, or L²-Ar²;    -   or R² and R³ together with the atoms to which R² and R³ are        bound form a substituted or unsubstituted ring;    -   each L¹ and L² is independently a linking group or a bond;    -   each Ar¹ is independently a substituted or unsubstituted aryl        group wherein Ar² is not substituted with an amide, amine,        nitro, imine, or an ester group; each Ar² is independently a        substituted or unsubstituted aryl group wherein Ar² is not        substituted with an amide, amine, nitro, imine, or an ester        group;    -   each A¹, A², A³, and A⁴ is independently C(R^(1a)),        C(R^(1a))(R^(1b)), N, or N(R^(1a));    -   each R^(1a) and R^(1b) is independently hydrogen, halogen,        hydroxyl, sulfhydryl, nitro, nitroso, cyano, azido, a sulfoxide        group, a sulfone group, a sulfonamide group, a sulfonic acid        group, an imine group, an acyl group, an acyloxy group, alkyl,        alkenyl, alkynyl, an alkoxy group, an ether group, a carboxylic        acid group, a carboxaldehyde group, an ester group, an amine        group, an amide group, a carbonate group, a carbamate group, a        thioether group, a thioester group, a thioacid group, aryl,        aryloxy, arylalkyl, arylalkoxy, heterocyclyl, heterocyclylalkyl,        heteroaryl, or heteroarylalkyl, any of which is substituted or        unsubstituted; and    -   each        is independently a single or double bond,        or a pharmaceutically-acceptable salt thereof, wherein the        compound is not:

Embodiment 141

The compound of embodiment 140, wherein when Ar¹ is phenyl brominated atone position, then Ar² is substituted on at least one position.

Embodiment 142

The compound of embodiment 140, wherein when Ar¹ is phenyl substitutedwith one methoxy group, then Ar² is substituted on at least oneposition.

Embodiment 143

The compound of embodiment 140, wherein Ar¹ is substituted, and Ar² issubstituted.

Embodiment 144

The compound of embodiment 140, wherein Ar¹ is unsubstituted, and Ar² isunsubstituted.

Embodiment 145

The compound of embodiment 134, wherein both L¹ and L² are independently

Embodiment 146

The compound of embodiment 139, wherein both Ar¹ and Ar² areindependently substituted with hydrogen, halogen, or alkyloxy.

Embodiment 147

The compound of embodiment 134, wherein each linking group isindependently alkylene, alkenylene, O, S, SO₂, CO, N₂, or a bond.

Embodiment 148

The compound of embodiment 134, wherein each

is independently chosen to provide an aromatic system.

Embodiment 149

The compound of any one of embodiments 140-149, wherein the compound is:

Embodiment 150

The compound of any one of embodiments 140-149, wherein the compound is:

Embodiment 151

The compound of any one of embodiments 140-149, wherein the compound is:

Embodiment 152

The compound of any one of embodiments 140-149, wherein the compound is:

Embodiment 153

The compound of any one of embodiments 140-149, wherein the compound is:

1-52. (canceled)
 53. A method of treating a condition, the methodcomprising administering to a subject in need thereof atherapeutically-effective amount of a compound and atherapeutically-effective amount of a second agent, wherein the compoundis of the formula:

wherein: R¹ is hydrogen or an ester group; R² is hydrogen, halogen, orL¹-Ar¹; R³ is hydrogen, halogen, or L²-Ar²; each L¹ and L² isindependently alkylene, alkenylene, O, S, SO₂, CO, N₂ or a bond; Ar¹ isa substituted or unsubstituted aryl group; Ar² is a substituted orunsubstituted aryl group wherein Ar² is not substituted with an amide,amine, nitro, imine, or ester group; each A¹, A², A³, and A⁴ isindependently C(R^(1a)), C(R^(1a))(R^(1b)), N, or N(R^(1a)); each R^(1a)and R^(1b) is independently hydrogen, halogen, hydroxyl, sulfhydryl,nitro, nitroso, cyano, azido, a sulfoxide group, a sulfone group, asulfonamide group, a sulfonic acid group, an imine group, an acyl group,an acyloxy group, alkyl, alkenyl, alkynyl, an alkoxy group, an ethergroup, a carboxylic acid group, a carboxaldehyde group, an ester group,an amine group, an amide group, a carbonate group, a carbamate group, athioether group, a thioester group, a thioacid group, aryl, aryloxy,arylalkyl, arylalkoxy, heterocyclyl, heterocyclylalkyl, heteroaryl, orheteroarylalkyl, any of which is substituted or unsubstituted; and each

is independently a single or double bond, or apharmaceutically-acceptable salt thereof, wherein the compound is not:


54. The method of claim 1, wherein the compound is of the formula:

wherein: R¹ is hydrogen; R² is hydrogen or L¹-Ar¹; R³ is L²-Ar²; andeach A¹, A², A³, and A⁴ is independently C(R^(1a)) or N, or apharmaceutically-acceptable salt thereof.
 55. The method of claim 53,further comprising irradiating the compound with light having awavelength of about 200 nm to about 800 nm.
 56. The method of claim 53,wherein the condition is an infection.
 57. The method of claim 56,wherein the infection is caused by a microbe.
 58. The method of claim57, wherein the microbe is a bacterium.
 59. The method of claim 57,wherein the microbe is a Gram-positive bacterium.
 60. The method ofclaim 57, wherein the microbe is a Gram-negative bacterium.
 61. Themethod of claim 57, wherein the microbe is a drug resistant bacterium.62. The method of claim 57, wherein the microbe is methicillin-resistantStaphylococcus aureus.
 63. The method of claim 57, wherein the microbeis Acinetobacter baumannii.
 64. The method of claim 53, wherein thecompound binds a biological structure.
 65. The method of claim 64,wherein the biological structure is an efflux pump.
 66. The method ofclaim 53, wherein the compound decreases an activity of a drugresistance mechanism in a microbe.
 67. The method of claim 53, whereinthe second agent is polymyxin B or a pharmaceutically-acceptable saltthereof.
 68. The method of claim 53, wherein the compound and the secondagent are administered in a common unit dosage form.
 69. The method ofclaim 53, wherein the administration is oral.
 70. The method of claim53, wherein the administration is intravenous.
 71. The method of claim53, wherein the administration is subcutaneous.
 72. The method of claim53, wherein the administration is topical.